U.S. patent application number 16/139831 was filed with the patent office on 2019-07-11 for oligonucleotide encoded chemical libraries.
This patent application is currently assigned to Plexium, Inc.. The applicant listed for this patent is Plexium, Inc.. Invention is credited to Andrew Boyd MACCONNELL, Joseph Franklin Rokicki, Michael VAN NGUYEN, Kandaswamy VIJAYAN.
Application Number | 20190210018 16/139831 |
Document ID | / |
Family ID | 65807199 |
Filed Date | 2019-07-11 |
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United States Patent
Application |
20190210018 |
Kind Code |
A1 |
VIJAYAN; Kandaswamy ; et
al. |
July 11, 2019 |
OLIGONUCLEOTIDE ENCODED CHEMICAL LIBRARIES
Abstract
This application provides a bead with a covalently attached
chemical compound and a covalently attached DNA barcode and methods
for using such beads. The bead has many substantially identical
copies of the chemical compound and many substantially identical
copies of the DNA barcode. The compound consists of one or more
chemical monomers, where the DNA barcode takes the form of barcode
modules, where each module corresponds to and allows identification
of a corresponding chemical monomer. The nucleic acid barcode can
have a concatenated structure or an orthogonal structure. Provided
are method for sequencing the bead-bound nucleic acid barcode, for
cleaving the compound from the bead, and for assessing biological
activity of the released compound.
Inventors: |
VIJAYAN; Kandaswamy; (San
Diego, CA) ; MACCONNELL; Andrew Boyd; (San Diego,
CA) ; Rokicki; Joseph Franklin; (Del Mar, CA)
; VAN NGUYEN; Michael; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Plexium, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
Plexium, Inc.
San Diego
CA
|
Family ID: |
65807199 |
Appl. No.: |
16/139831 |
Filed: |
September 24, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62562905 |
Sep 25, 2017 |
|
|
|
62562912 |
Sep 25, 2017 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C40B 50/16 20130101;
C12Q 2600/16 20130101; C40B 30/06 20130101; C40B 30/04 20130101;
C40B 50/14 20130101; C40B 50/04 20130101; C12N 15/1065 20130101;
C12N 15/1034 20130101; C12Q 1/6876 20130101; B01L 2300/0829
20130101; B01L 3/50853 20130101; C12Q 1/6869 20130101; C12N 15/1065
20130101; C12Q 2563/179 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; C12N 15/10 20060101 C12N015/10; C40B 30/04 20060101
C40B030/04 |
Claims
1. A system for screening chemical compounds, comprising: (a) A
picowell array plate comprising a plurality of picowells, wherein
each picowell has a top aperture that defines an opening at the top
of the picowell, a bottom that is defined by a floor, wherein the
top aperture is separated by a wall from the floor, and wherein the
wall resides in between the top aperture and the floor, (b) A bead
disposed in a picowell, wherein the bead comprises a plurality of
substantially identical bead-bound DNA barcodes, and a plurality of
substantially identical bead-bound compounds, (c) Wherein the bead
comprises a bead-bound DNA barcode that takes the form of either a
concatenated DNA barcode or an orthogonal DNA barcode, and wherein
if the DNA barcode takes the form of a concatenated DNA barcode the
concatenated DNA barcode is made by a method that uses one or both
of: (i) Uses click chemistry, or (ii) Uses a repeating cycle of
steps, wherein the repeating cycle of steps comprises using a
splint oligonucleotide (splint oligo) that is capable of
hybridizing to a partially made bead-bound DNA barcode, and wherein
the hybridizing is mediated by an annealing site on the splint
oligo and a corresponding, complementary annealing site in the
partially made bead-bound DNA barcode, wherein the annealed splint
oligo is used as a template for extending the partially made DNA
barcode using DNA polymerase, and wherein the splint oligo contains
bases that are complementary to a DNA barcode module that is to be
polymerized to the partially made bead-bound DNA barcode, and
wherein the splint oligo also contains bases that are complementary
to an annealing site that is to be polymerized to the partially
made bead-bound DNA barcode, and (d) Wherein each one of the
plurality of substantially identical bead-bound compounds comprises
one or more chemical library monomers, and wherein each bead-bound
DNA barcode module identifies a corresponding chemical library
monomer, wherein the term "compound" is used to refer to a
completed product that comprises one or more chemical library
members, and wherein the completed DNA barcode identifies the
compound.
2. The system of claim 1, further comprising an oligonucleotide
sequencing primer that is capable of guiding the sequencing of one
or more DNA barcode modules that is comprised by a bead-bound DNA
barcode, where optionally the system comprises a DNA sequencing
machine, and where the DNA sequencing machine is not a
luminescence-based sequencer and not a pH-based DNA sequencing
machine.
3. The system of claim 1, further comprising a plurality of
spherical caps, wherein each cap is capable of fitting into the
aperture of a picowell wherein the aperture is circular, and each
cap is capable of minimizing or preventing evaporation of fluid
that is inside of the picowell, and each cap is capable of
minimizing or preventing leakage of fluid that is inside of the
picowell.
4. The system of claim 1, wherein the at least one bead disposed in
the at least one picowell comprises at least one response capture
element that is coupled to said at least one bead.
5. The system of claim 1, wherein the at least one of the a bead
disposed in a picowell comprises at least one response capture
element that is coupled to said at least one bead, wherein the at
least one response capture element comprises: (a) Poly(dT); (b) An
exon-targeting RNA probe; (c) An antibody; or (d) An aptamer.
6. The system of claim 1, wherein the DNA barcode is either a
concatenated DNA barcode or an orthogonal DNA barcode, and wherein
the DNA barcode comprises one or more DNA barcode modules, wherein
each of the one or more DNA barcode modules encodes information
that identifies a chemical library monomer, and wherein the
concatenated DNA barcode or the orthogonal DNA barcode further
includes one or both of: (a) One or more functional nucleic acids;
and (b) One or more nucleic acids that encode information of a type
other than the identity of a chemical library monomer.
7. The system of claim 1, wherein the bead-bound concatenated DNA
barcode comprises: (i) a 1.sup.st DNA barcode module; or (i) a
1.sup.st DNA barcode module, a 1.sup.st annealing site, and a
2.sup.nd DNA barcode module; or (ii) a 1.sup.st DNA barcode module,
a 1.sup.st annealing site, a 2.sup.nd DNA barcode module, a
2.sup.nd annealing site, and a 3.sup.rd DNA barcode module; or
(iii) a 1.sup.st DNA barcode module, a 1.sup.st annealing site, a
2.sup.nd DNA barcode module, a 2.sup.nd annealing site, a 3.sup.rd
DNA barcode module, a 3.sup.rd annealing site, and a 4.sup.th DNA
barcode module; or (iv) a 1.sup.st DNA barcode module, a 1.sup.st
annealing site, a 2.sup.nd DNA barcode module, a 2.sup.nd annealing
site, a 3.sup.rd DNA barcode module, a 3.sup.rd annealing site, a
4.sup.th DNA barcode module, a 4.sup.th annealing site, and a
5.sup.th DNA barcode module; or (v) a 1.sup.st DNA barcode module,
a 1.sup.st annealing site, a 2.sup.nd DNA barcode module, a
2.sup.nd annealing site, a 3.sup.rd DNA barcode module, a 3.sup.rd
annealing site, a 4.sup.th DNA barcode module, a 4.sup.th annealing
site, a 5.sup.th DNA barcode module, a 5.sup.th annealing site, and
a 6.sup.th DNA barcode module.
8. The system of claim 1, wherein the bead comprises a DNA barcode
that is an orthogonal DNA barcode, wherein the bead comprises an
external surface, and wherein the orthogonal DNA barcode comprises:
(a) A first nucleic acid that comprises a first DNA barcode module
and an annealing site for a sequencing primer, wherein the first
nucleic acid is coupled to the bead at a first position, (b) A
second nucleic acid that comprises a second DNA barcode module and
an annealing site for a sequencing primer, wherein the second
nucleic acid is coupled to the bead at a second position, and (c) A
third nucleic acid that comprises a third DNA barcode module and an
annealing site for a sequencing primer, wherein the second nucleic
acid is coupled to the bead at a third position, and wherein the
first, second, and third position on the bead are each located at
different location on the bead's external surface.
9. The system of claim 1, wherein the concatenated DNA barcode is
made by a method that uses: (i) Both click chemistry and the
repeating cycle of steps that uses the splint oligo; (ii) Both
click chemistry and chemical methods that are not click chemistry
methods; (iii) Only click chemistry; or (iv) Only the repeating
cycle of steps that uses the splint oligo.
10. The system of claim 1, wherein each of the plurality of
substantially identical bead-bound compounds is coupled to the bead
by way of a cleavable linker, or by way of a cleavable linker that
is a light-cleavable linker, or by way of a non-cleavable
linker.
11. The system of claim 1, wherein the at least one bead comprises
grafted copolymers consisting of a low crosslinked polystyrene
matrix on which polyethylene glycol (PEG) is grafted.
12. The system of claim 1, wherein at least one picowell comprises
at least one cell, wherein the plurality of substantially identical
bead-bound compounds are bound to the at least one bead by way of a
cleavable linker, and wherein cleaving the cleavable linker
releases the bead-bound compound from the bead to produce a
released compound, and wherein the released compound is capable of
contacting the at least one cell, and wherein the at least one cell
is: (i) a mammalian cell that is not a cancer cell, (ii) a
mammalian cancer cell, (iii) a dead mammalian cell, (iv) an
apoptotic mammalian cell, (v) a necrotic mammalian cell, (vi) a
bacterial cell, (vii) a plasmodium cell, (vii) a cell that is
metabolically active but has a cross-linked genome and is unable to
undergo cell division, or (ix) a mammalian cell that is infected
with a virus.
13. The system of claim 1, wherein each picowell has a top aperture
that defines an opening at the top of the picowell, a bottom that
is defined by a floor, wherein the top aperture is separated from
the floor, and wherein a wall resides in between the top aperture
and the floor, and wherein the aperture is round, wherein the floor
is round, and wherein the wall takes the form of a truncated cone,
and wherein the aperture has a first diameter, the floor has a
second diameter, and wherein the first diameter is greater than the
second diameter.
14. The system of claim 1, wherein each picowell has a top aperture
that defines an opening at the top of the picowell, a bottom that
is defined by a floor, wherein the top aperture is separated from
the floor, and wherein a wall resides in between the top aperture
and the floor, and wherein the aperture is round, wherein the floor
is round, and wherein the wall takes the form of a truncated cone,
and wherein the aperture has a first diameter, the floor has a
second diameter, and wherein the first diameter is greater than the
second diameter, further comprising a cap that snuggly fits into
the aperture, wherein the aperture is comprised by a polymer having
a greater durometer (harder) and wherein the cap is made of a
polymer having a lesser durometer (softer), and wherein the
relative durometers of the cap and aperture allow the cap to be
reversibly and snuggly fit into the aperture, and wherein the cap
is: (i) a cap intended only to plug the picowell and prevent
leakage, (ii) a cap that is a passive cap and that is capable of
absorbing metabolites that are released by a cell, in the situation
where a cell in a cell medium is cultured in the picowell, (iii) a
cap that is an active cap, and that takes the form of a bead that
comprises a plurality of essentially identical compounds, and
wherein each of the plurality of essentially identical compounds is
coupled to the bead with a cleavable linker; (iv) a cap that is an
active cap, and that takes the form of a bead that comprises a
plurality of identical reagents, and wherein each of the plurality
of essentially identical reagents is coupled to the bead with a
cleavable linker.
15. The system of claim 14, further comprising at least one
spherical cap.
16. The system of claim 14, further comprising at least one
non-spherical cap.
17. The system of claim 1, wherein the DNA barcode comprises one or
more nucleic acids that do not encode any chemical monomer but
instead identify one or more of: (a) The class of chemical
compounds that is cleavably attached to the bead; (b) The step in a
multi-step pathway of organic synthesis, wherein a bead-bound
nucleic acid corresponds to a given chemical monomer that is used
to make a bead-bound compound, and wherein the bead-bound nucleic
acid that corresponds to a given chemical monomer identifies that
chemical monomer; (c) The date that the bead-bound compound was
synthesized; (d) The disease that the bead-bound compound is
intended to treat; (e) The cellular event that the bead-bound
compound is intended to stimulate or inhibit; or (f) The reaction
conditions that were used to couple a given chemical library
monomer to the bead.
18. The system of claim 1, wherein there does not exist any
headpiece that links any of the bead-bound compounds to any of the
bead-bound DNA barcodes.
19. The system of claim 1, wherein the concatenated DNA barcode
comprises at least one nucleic acid that is a DNA barcode module,
and at least one functional nucleic acid that: (a) Is capable of
being used as an annealing site for a sequencing primer, (b) Is
capable of forming a hairpin structure, and wherein the hairpin
structure comprises a sequencing primer, an annealing site for the
sequencing primer, and a bend in the hairpin structure wherein the
bend is 5-prime to the sequencing primer and is 3-prime to the
annealing site for the sequencing primer, or (c) Is a spacer
nucleic acid.
20. The system of claim 1, wherein the orthogonal DNA barcode
contains a plurality of DNA barcode modules, wherein each of the
DNA barcode modules is coupled to a different site on the bead
either directly or via a linker, and wherein each of the plurality
of DNA barcode modules contains at least one functional nucleic
acid that is: (a) Capable of being used as an annealing site for a
sequencing primer, (b) Capable of forming a hairpin structure, and
wherein the hairpin structure comprises a sequencing primer, an
annealing site for the sequencing primer, and a bend in the hairpin
structure wherein the bend is 5-prime to the sequencing primer and
is 3-prime to the annealing site for the sequencing primer, or (c)
A spacer nucleic acid.
21. A method for controlling the concentration of a compound in a
solution that resides in a picowell, wherein the method is applied
to a bead-bound compound in a picowell, wherein the picowell
contains a solution, and wherein the bead-bound compound is coupled
to the bead by way of a cleavable linker, the method comprising:
(a) The step of exposing the bead-bound compound to a condition
that effects cleavage of the cleavable linker, wherein the
condition comprises light that is capable of cleaving the cleavable
linker, (b) The step of allowing release of the bead-bound compound
from the bead to generate a released compound, wherein release is
followed by diffusion or dispersion of the released compound in the
solution to result in a substantially uniform concentration of the
compound in the solution, (c) The step of adjusting the condition
to produce a determined concentration of the substantially uniform
concentration, wherein the determined concentration is made with
regard to the concentration of a released fluorophore that is
released by from a bead-bound release-monitor.
22. The method of claim 21, wherein the condition is adjusted by
adjusting one or more of the wavelength of the light, the intensity
of the light, and by the duration of light exposure and wherein,
optionally: (i) The concentration of a released fluorophore that is
released from a bead-bound release-monitor is determined at the
same time as effecting release of the bead-bound compound from the
bead to generate a released compound, or (ii) The concentration of
a released fluorophore that is released from a bead-bound
release-monitor is determined at a time substantially before
effecting release of the bead-bound compound from the bead to
generate a released compound.
23. A cap in combination with a picowell plate that comprises a
plurality of picowells, wherein the cap is capable of use with said
picowell plate, wherein each of the plurality of picowells is
definable by an aperture, a floor, and a wall, wherein the wall is
defined by the aperture on top and the floor on the bottom, and
wherein the aperture is round, wherein the floor is round, and
wherein the wall takes the form of a surface of a truncated cone,
and wherein the aperture has a first diameter, the floor has a
second diameter, and wherein the first diameter is greater than the
second diameter, wherein the cap is a spherical cap that is capable
of snuggly fitting into the aperture, wherein the aperture is
comprised by a polymer having a greater durometer (harder) and
wherein the cap is made of a polymer having a lesser durometer
(softer), and wherein the relative durometers of the cap and
aperture allow the spherical cap to be reversibly and snuggly fit
into the aperture, and wherein the cap is: (i) capable of plugging
the picowell and preventing leakage, (ii) a passive cap and that is
capable of absorbing metabolites that are released by a cell, in
the situation where a cell in a cell medium is cultured in the
picowell, (iii) an active cap that takes the form of a bead that
comprises a plurality of essentially identical compounds, and
wherein each of the plurality of essentially identical compounds is
coupled to the bead with a cleavable linker, wherein at least one
of the plurality of picowells contains an aqueous medium, and
wherein cleavage of the cleavable linker releases at least some of
the plurality of essentially identical compounds from the bead into
the aqueous medium.
24. A system comprising a picowell array plate comprising an upper
generally planar surface, a plurality of picowells, wherein each
picowell has a top aperture that defines an opening at the top of
the picowell, a bottom that is defined by a floor, wherein the top
aperture is separated by a wall from the floor, and wherein the
wall resides in between the top aperture and the floor, and
optionally, a bead disposed in at least one of said plurality of
picowells, wherein the bead comprises a plurality of substantially
identical bead-bound DNA barcodes, and a plurality of substantially
identical bead-bound compounds, wherein the picowell array plate
further comprises a mat that is capable of securely covering the
opening at the top of at least one or all of the plurality of
picowells, or that is actually securely covering the opening at the
top of at least one or all of the plurality of picowells, wherein
the securely covering is reversible, wherein the mat optionally
comprises one or all of: (a) An absorbant surface that, when
positioned in contact with the upper generally planar surface of
the picowell array plate, is capable of absorbing any metabolites,
biochemicals, or proteins that may be comprised by one or more of
the plurality of picowells, (b) An adhesive surface that is capable
of maintaining reversible adhesion to the top generally planar
surface of the picowell array plate.
25. A method for determining a signal from an assay and a
sequencing readout on a bead, thereby identifying one or more
compounds of interest from the assay, comprising the steps: (a)
providing a plurality of beads, wherein each bead comprises a
plurality of compounds attached to the bead that are related
substantially to each other, and a plurality of oligonucleotides,
wherein the plurality of oligonucleotides attached to each bead
identify the plurality of compounds attached to the same bead; (b)
performing the assay involving the plurality of compounds attached
to the beads; (c) determining at least one signal that reflects the
performance of the compounds in the assay of step b; (d) sequencing
the plurality of oligonucleotides attached to the beads, without
removing the oligonucleotides from the bead, thereby determining a
sequencing readout for each bead; and (e) identifying the compounds
attached to the bead by the sequencing readout of step d and
relating it to the assay performance contained in the determined
signal of step c, wherein beads having a signal from the assay and
the sequencing readout identify the compound of interest.
26. A method for screening a compound library for compounds having
desired properties, comprising: (a) providing a plurality of beads,
wherein each bead comprises a plurality of oligonucleotides
attached to the bead surface and a plurality of substantially
related compounds attached to the bead surface, and wherein the
sequence of the oligonucleotides attached to the beads encodes the
identity of the plurality of substantially related compounds
attached to the bead surface; (b) incorporating the plurality of
beads in an assay for desired properties of compounds in the
compound library; (c) capturing a signal from at least one bead,
wherein the signal reflects the performance of the compounds on the
bead in the assay; (d) sequencing the plurality of oligonucleotides
attached to the at least one bead for which assay signal was also
captured, without removing the oligonucleotides from the bead; and
(e) identifying at least one compound from the sequencing readout
of step (d) and relating it to its corresponding assay performance
captured in the signal of step (c).
27. The method of claim 26, wherein each bead comprises a different
plurality of oligonucleotides and a different plurality of
substantially related compounds.
28. The method of claim 25, wherein the plurality of
oligonucleotides is a plurality of DNA oligonucleotides.
29. The method of claim 25, wherein the plurality of compounds is
attached to the bead surface by joining multiple compound building
blocks in tandem, wherein all the compound building blocks together
make up the compound.
30. The method of claim 29, wherein each DNA module and each
compound building block are assembled sequentially and
alternatively.
31. The method of claim 25, wherein each compound in the plurality
of identical compounds is attached to the bead surface by way of a
cleavable linker.
32. The method of claim 31, wherein the cleavable linker is a
photocleavable linker, a protease cleavable linker, or an acid
cleavable linker.
33. The method of claim 25, wherein the compounds are cleaved from
the bead surface after step (a) and prior to step (d).
34. The method of claim 25, wherein the signal that reflects the
desired property of the compound is a fluorescent signal.
35. The method of claim 25, wherein the size of each bead is
between 1 .mu.m and 100 .mu.m.
36. The method of 35, wherein the size of each bead is between 1
.mu.m and 10 .mu.m.
37. The method of 36, wherein the size of each bead is about 3
.mu.m.
38. The method of claim 25, wherein the method further comprises
identifying a target candidate in a plurality of potential targets,
and wherein the compound having the desired property binds to the
target candidate.
39. The method of claim 38, wherein step (b) comprises incubating
the plurality of beads in the plurality of potential targets.
40. The method of claim 38, wherein the potential targets are
proteins or nucleic acids.
41. The method of claim 25, wherein the sequencing is performed by
single-molecule real-time sequencing, ion semiconductor sequencing,
pyrosequencing, sequencing by synthesis, sequencing by bridge
amplification, sequencing by ligation, nanopore sequencing, chain
termination sequencing, massively parallel signature sequencing,
polony sequencing, heliscope single molecule sequencing, shotgun
sequencing, SOLiD sequencing, Illumina sequencing, tunneling
currents DNA sequencing, sequencing by hybridization, sequencing
with mass spectrometry, microfluidic Sanger sequencing, and
oligonucleotide extension sequencing.
42. A method for screening a compound library for compounds having
desired properties, comprising: (a) providing a plurality of beads,
wherein each bead comprises a plurality of oligonucleotides
attached to the bead surface and a plurality of substantially
related compounds attached to the bead surface, and wherein the
sequence of the oligonucleotides attached to the beads encodes the
synthesis history of the plurality of substantially related
compounds attached to the bead surface; (b) incorporating the
plurality of beads in an assay for desired properties of compounds
in the compound library; (c) capturing a signal from at least one
bead, wherein the signal reflects the performance of the compounds
on the bead in the assay; (d) sequencing the plurality of
oligonucleotides attached to the at least one bead for which assay
signal was also captured, without removing the oligonucleotides
from the bead; and (e) identifying at least one compound from the
sequencing readout of step (d) and relating it to its corresponding
assay performance captured in the signal of step (c).
43. The method of claim 42, wherein the assay comprises a binding
assay.
44. The method of claim 42, wherein the assay comprises an activity
assay.
45. The method of claim 42, wherein the assay comprises a
competitive binding assay or a competitive inhibition assay.
46. The method of claim 42, wherein the assay comprises interaction
of untethered compounds with other assay reagents, wherein the
untethered compounds are compounds released from the bead
surface.
47. The method of claim 45, wherein the compounds are released by
cleaving a cleavable linker that connects the compounds to the
beads.
48. The method of claim 42, wherein the assay occurs in a plurality
of confined volumes, wherein nominally one bead is dispersed per
confined volume.
49. The method of claim 48, wherein the confined volume comprises
an aqueous droplet.
50. The method of claim 49, wherein the aqueous droplet is
suspended in an oil medium or a hydrophobic liquid medium.
51. The method of claim 48, wherein the confined volume comprises a
picowell.
52. The method of claim 50, wherein the picowells are organized in
a regular array.
53. The method of claim 51, wherein the plurality of confined
volumes are organized in a regular array.
54. The method of claim 48, wherein the confined volume comprises a
layer of adherent aqueous medium around the bead, wherein the bead
is suspended in a hydrophobic medium.
55. The method of claim 42, wherein the assay reagents are washed
away before sequencing the oligonucleotides.
56. The method of claim 42, wherein the sequencing step (d) is
performed before the assay step (b).
57. The method of claim 56, wherein the oligonucleotides on the
beads are removed after the sequencing step, but before the assay
step.
58. The method of claim 57, wherein the removing of the
oligonucleotide comprises an enzymatic digestion, a chemical
cleavage, a thermal degradation or a physical shearing.
59. The method of claim 43, wherein the binding assay comprises
binding of RNA molecules to the beads.
60. The method of claim 43, wherein the signal from the bead
comprises sequencing of the bound RNA molecules.
61. The method of claim 42, wherein the binding assay comprises a
fluorescently labeled binding assay, wherein the molecules binding
to the compounds on the beads comprise fluorophores.
62. The method of claim 42, wherein the binding assay comprises
nucleic-acid labeled binding assay, wherein the molecules binding
to the compounds on the beads comprise nucleic-acid tags, wherein
further the signal from the assay comprises sequencing of the
nucleic acid tags attached to the molecules binding to the
compounds on the beads.
63. The method of claim 42, wherein the desired properties include
one or more of: (i) Inhibiting or stimulating the catalytic
activity of an enzyme, (ii) Stimulating Th1-type immune response,
as measurable by cell-based assays or by in vivo assays, (iii)
Stimulating Th2-type immune response, as measurable by cell-based
assays or by in vivo assays, (iv) Inhibiting Th1-type immune
response, as measurable by cell-based assays or by in vivo assays,
(v) Inhibiting Th2-type immune response, as measurable by
cell-based assays or by in vivo assays, (vi) Stimulating or
inhibiting ubiquitin-mediated degradation of a protein, as
measurable by purified proteins, by cell-based assays, or by in
vivo assays.
64. A system for screening a compound library for a compound having
a desired activity, comprising: (a) a sample compartment for
receiving a plurality of compound-attached, oligonucleotide-encoded
beads; (b) a plurality of encapsulation compartments within the
sample compartment, each encapsulation compartment nominally
comprising a single bead dispersed in an assay medium, wherein
further the assay medium comprises reagents whose interaction with
the compounds on the beads is being assayed resulting in a
measurable signal; (c) a detector for measuring signals; (d) a
sequencing platform; and (e) a user interface for receiving one or
more commands from a user.
65. The system of claim 64, wherein the encapsulation compartment
comprises a liquid droplet.
66. The system of claim 64, wherein the encapsulation compartment
comprises a picowell.
67. The system of claim 64, wherein further the encapsulation
compartment comprises assay reagents.
68. The system of claim 64, wherein the detector comprises an
optical detector.
69. The system of claim 64, wherein the sequencer comprises the
optical detector.
70. The system of claim 1, further comprising a plurality of caps,
each cap capable of fitting into the opening of a different
picowell, and each cap capable of minimizing or preventing
evaporation of fluid that is inside of the picowell, and each cap
is capable of minimizing or preventing leakage of fluid that is
inside of the picowell.
Description
CROSS REFERENCE TO RELATED CASES
[0001] This application claims the benefit of, and priority to,
U.S. Provisional Patent Application Ser. No. 62/562,905 filed Sep.
25, 2017, and U.S. Provisional Patent Application Ser. No.
62/562,912, also filed Sep. 25, 2017, the contents of which are
incorporated herein by reference in its entirety.
FIELD OF THE DISCLOSURE
[0002] The disclosure relates to high-throughput screening using a
library of compounds, where the compounds are bound to beads, or
contained within beads, each bead containing multiple copies of one
kind of compound, where further, the bead also contains DNA tags
that encode the identity or synthetic history of the compound that
is contained in or on the bead. The disclosure so relates to
high-throughput assays performed in picowells, where the picowells
contain compound-laden beads and assay materials. The disclosure
further relates to releasing the bead-bound compounds and screening
them for biological activity. Broadly, the disclosure contemplates
assays where beads are used as delivery-vehicles for compounds, and
methods for creating such compound-laden beads.
[0003] The disclosure relates bead-bound compounds, where each
compound is made of one or more monomers belonging to a chemical
library. The disclosure also relates to bead-bound DNA barcodes,
that is, to nucleic acids where the sequence of each nucleic acid
is a code (not related to the genetic code) refers to one
particular chemical library monomer. The disclosure further relates
to releasing the bead-bound compounds and then screening the
released compounds for biological activity.
[0004] The disclosure also pertains generally to methods for
perturbing a cell, or a few cells, with a dose-controlled compound,
and analyzing the change in the state of the cell by RNA and/or
protein analysis. The methods disclosed herein could be applied at
the single-cell level, or to a plurality of cells, for the purpose
of high throughput screening, target discovery, or diagnostics, and
other similar applications.
BACKGROUND OF THE DISCLOSURE
[0005] Combinatorial chemistry, for example, involving
split-and-pool chemistry, can be used for synthesizing large
amounts of compounds. Compounds made in this way find use in the
field of medicinal chemistry, where the compounds can be screened
for various biochemical activities. These activities include
binding to one or more proteins, where the proteins are known at
the time the screening test is performed. Alternatively, the
proteins that are bound by a compound being tested are identified
only after a binding event is detected. Compounds can also be
screened for their activity of inhibiting or activating a known
protein (this is not merely screening for a "binding" activity).
Alternatively, compounds can be screened for their activity of
inhibiting or activating a cellular function, and where the
molecular targets are not known to the researcher at the time of
screening.
[0006] The screening of compounds, such as compounds belonging to a
huge library of chemicals made by split-and-pool methods, can be
facilitated by conducting screening with an array of many thousands
of microwells, nanowells, or picowells. Moreover, screening can be
facilitated by providing a different compound to each picowell by
way of a bead, and where each bead contains hundreds of copies of
the same compound, and where the same bead also contains hudreds of
copies of a "DNA barcode" that can be used to identify the compound
that is attached to the same bead. Moreover, screening of compounds
is further facilitated by using cleavable linkers, where the
cleavable linker permits controlled release of the compound from
the bead, and where the released compound is then used for
biochemical assays or cell-based assays in the same picowell.
[0007] Assaying compounds in very small, confined volumes, such as
droplets, picowells or microfluidic environments is broadly
beneficial, for instance, due to the low volumes of assay reagents
needed, and therefore need not be limited to combinatorially
generated compounds. Any method that can load compounds onto beads,
that also allows the compounds to be eluted off the beads at a
later time, may be used for delivering bead-bound compounds to
assays in small, confined volumes. The addition of nucleic acid
barcodes to the beads allows the identity of the compound present
within the beads to be carried along to the assay volume. In his
manner, very high throughput assays may be performed without
needing robotics or spatial indexing of compounds within microtiter
plates. Millions to billions of compounds may be held within one
small vial, the identity of the compounds tagged on the same bead
(with DNA) that contains each individual compound.
[0008] A common method for drug discovery involves picking a target
of interest and monitoring the interaction of the target protein or
enzyme with a large library of chemical compounds. In many cases, a
large number of initial hits are found toxic to the body or cross
reactive with other proteins in the body, rendering the
target-based selection an inefficient method for drug screening.
The need for a pre-selected target is also an inherent limitation,
since it requires the biological underpinning of disease to be
well-known and understood. Screening compounds against an entire
organism is a difficult, expensive, and very low-throughput
task.
[0009] Conventional phenotypic screening on cells has involved
creating models of diseased-state cells, contacting the cells with
various drug libraries, and monitoring if the disease phenotype is
corrected by a measurable assay. Such screening methods are called
phenotypic screening, as the underlying biological mechanism is not
necessarily understood at the beginning, but a measurable,
phenotypic change that is indicative of a curative response is
considered the relevant metric. A vast number of cell lines and
disease models reflecting various baseline and diseased cell states
are available today. Also available are larger numbers of compound
libraries and biological drugs candidates. The obvious screening
campaign combining different cell models with different drug
candidates to look for phenotypic responses is fraught with
technical limitations as assays are limited to microtiter plate
formats and imaging modalities, both of which are severely limited
in throughput.
[0010] One method to overcome throughput limitations is to adopt
high-throughput single-cell screening approaches to drug discovery
(see, e.g., Heath et al., Nat Rev Drug Discov. 15:204-216, 2016).
In these approaches, single cells are separated and isolated into
compartments where individual assays can be performed on each of
the cells. Genomic analysis via mRNA sequencing of the single
cells, e.g., using droplet encapsulation, is a popular method that
reveals intricate details that are hidden in ensemble measurements
(see, e.g., Macosko et al., Cell 161:1202-1214, 2015 and Ziegenhain
et al., Mol Cell 65:631-643, 2017, the disclosures of which are
incorporated herein by reference in their entireties). Present
state of the art single-cell analysis platforms have enabled
quantitation of mRNA transcripts with single-cell resolution to
characterize and fingerprint cells based on their transcriptional
state. This approach allows for comparison between tissue samples,
extracted from a subject or prepared in an experiment, and
examining single-cell transcription, and therefore, protein
expression states. The measurements of single-cell mRNA by
transcriptome sequencing and profiling are important approaches to
investigate molecular mechanisms of not only genealogic phenotypes
of cells during disease progression, but also drug efficacy,
resistances, and discovery of therapeutic targets (see, e.g., Chu
et al., Cell Biol and Toxicol 33:83-97, 2017, Wang, Cell Biol
Toxicol 32:359-361, 2016, and Wang et al., Cell Biol Toxicol
33:423-427, 2017). The application of single-cell RNA sequencing
has been used to define intercellular heterogeneity, evidenced by
transcriptomic cell-to-cell variation, which is extremely relevant
to drug efficacy and specificity, transcriptional stochasticity,
transcriptome plasticity, and genome evolution. Encapsulation in
picowells has also been demonstrated (see, e.g., Gierahn et al.,
Nat Methods 14:395-398, 2017). Single cell protein measurements are
also possible using similar isolation methods (Butnik et al.,
BioRxiv, January 2017, Su et al., Proteomics 17:3-4, 2017).
[0011] Despite the rapid rise in high-throughput single-cell
RNA-sequencing (RNA-seq) methods, including commercialized versions
of automated platforms such as the Fluidigm C1, 10.times.Genomics
or 1CellBiO systems, the application of single-cell RNA profiling
for target agnostic high-throughput drug screening and target
discovery is constrained by the lack of methods that can
efficiently partition different drugs to different cells. While
incubating cells or tissues under different perturbations within
well plates, followed by single-cell analysis and comparisons
between transcript profiles can be done, the number of drugs that
can be examined is limited by the plate capacity. Further, the need
to prepare barcoded mRNA from each sample in isolation and then
perform comprehensive RNA profiles for every sample, creates a
major bottleneck, as well.
SUMMARY OF THE DISCLOSURE
[0012] Briefly stated, the present disclosure provides a system for
screening chemical compounds, comprising: (a) A picowell array
plate comprising a plurality of picowells, wherein each picowell
has a top aperture that defines an opening at the top of the
picowell, a bottom that is defined by a floor, wherein the top
aperture is separated from the floor, and wherein a wall resides in
between the top aperture and the floor; (b) A bead disposed in a
picowell, wherein the bead comprises a plurality of substantially
identical bead-bound DNA barcodes, and a plurality of substantially
identical bead-bound compounds, (c) Wherein the bead comprises a
bead-bound DNA barcode that takes the form of either a concatenated
DNA barcode or an orthogonal DNA barcode, and wherein if the DNA
barcode takes the form of a concatenated DNA barcode the
concatenated DNA barcode is made by a method that: (i) Uses click
chemistry, or (ii) Uses a repeating cycle of steps, wherein the
repeating cycle of steps comprises using a splint oligonucleotide
(splint oligo) that is capable of hybridizing to a partially made
bead-bound DNA barcode, and wherein the hybridizing is mediated by
an annealing site on the splint oligo and a corresponding,
complementary annealing site in the partially made bead-bound DNA
barcode, wherein the annealed splint oligo is used as a template
for extending the partially made DNA barcode using DNA polymerase,
and wherein the splint oligo contains bases that are complementary
to a DNA barcode module that is to be polymerized to the partially
made DNA barcode, (d) Wherein each one of the plurality of
substantially identical bead-bound compounds comprises one or more
chemical library monomers, and wherein each bead-bound DNA barcode
module identifies a corresponding chemical library monomer, wherein
the term "compound" is used to refer to a completed product that
comprises one or more chemical library members, and wherein the
completed DNA barcode identifies the compound.
[0013] The floor of a microwell, nanowell, or picowell, need not be
flat. The floor may be curved as in the manner of the bottom of a
glass test tube or metal centrifuge tube. Also, the floor may be
conical-shaped, as in conical centrifuge tubes. The floor may be
flat but with notches, for example, notches that facilitate motion
of an assay solution or cell culture solution in the vicinity of
the bottom of any bead that is sitting in the picowell. In
flat-floor embodiments, the present system and methods can require
a flat floor.
[0014] The concatenated DNA barcode can be made entirely by methods
of organic chemistry, for example, by click chemistry. Also, the
orthogonal DNA barcode can be made entirely by methods of organic
chemistry, for example, comprising click chemistry.
[0015] What is also provided is the above system, further
comprising a plurality of caps, each cap capable of fitting into
the opening of a different picowell, and each cap capable of
minimizing or preventing evaporation of fluid that is inside of the
picowell, and each capable of minimizing or preventing leakage of
fluid that is inside of the picowell.
[0016] Moreover, what is embraced is the above system, wherein the
concatenated DNA barcode is made by a method that uses: (i) Both
click chemistry and the repeating cycle of steps that uses the
splint oligo; (ii) Both click chemistry and chemical methods that
are not click chemistry methods; (iii) Only click chemistry; or
(iv) Only the repeating cycle of steps that uses the splint oligo.
For this particular embodiment the "concatenated DNA barcode" in
question does not include any chemical coupler that is used to
couple a nucleic acid directly to the bead.
[0017] In a spherical cap embodiment, what is provided is the above
system, further comprising a plurality of spherical caps, wherein
each cap is capable of fitting into the aperture of a picowell
wherein the aperture is circular, and each cap is capable of
minimizing or preventing evaporation of fluid that is inside of the
picowell, and each cap is capable of minimizing or preventing
leakage of fluid that is inside of the picowell.
[0018] In a response element embodiment, what is provided is the
above system, wherein the at least one bead disposed in the at
least one picowell comprises at least one response capture element
that is coupled to said at least one bead. Also, what is
contemplated is the above system, wherein the at least one bead
disposed in at least one picowell comprises at least one response
capture element that is coupled to said at least one bead, wherein
the at least one response capture element comprises: (a) Poly(dT)
or (b) An exon-targeting RNA probe.
[0019] Also contemplated is the above system, wherein the DNA
barcode is either a concatenated DNA barcode or an orthogonal DNA
barcode, and wherein the DNA barcode comprises one or more DNA
barcode modules, wherein each of the one or more DNA barcode
modules encodes information that identifies a chemical library
monomer, and wherein the concatenated DNA barcode or the orthogonal
DNA barcode further includes one or both of: (a) One or more
functional nucleic acids; and (b) One or more nucleic acids that
encode information of a type other than the identity of a chemical
library monomer.
[0020] The following discloses "consists of only" embodiments and
"comprises" embodiments, as it applies to the number of bead-bound
DNA barcode modules that make up a DNA barcode. What is provided is
embodiments where the DNA barcode consists of only one DNA barcode
module, or only two DNA barcode modules, or contains only three DNA
barcode modules, or only four DNA barcode modules, and so on, or
where the DNA barcode comprises at least one DNA barcode module, or
comprises at least two DNA barcode modules, or comprises at least
three DNA barcode modules, or comprises at least four DNA barcode
modules, and so on,
[0021] What is also embraced, is a system wherein the bead-bound
concatenated DNA barcode comprises: (i) a 1.sup.st DNA barcode
module; or (i) a 1.sup.st DNA barcode module, a 1.sup.st annealing
site, and a 2.sup.nd DNA barcode module; or (ii) a 1.sup.st DNA
barcode module, a 1.sup.st annealing site, a 2.sup.nd DNA barcode
module, a 2.sup.nd annealing site, and a 3.sup.rd DNA barcode
module; or (iii) a 1.sup.st DNA barcode module, a 1.sup.st
annealing site, a 2nd DNA barcode module, a 2nd annealing site, a
3.sup.rd DNA barcode module, a 3.sup.rd annealing site, and a
4.sup.th DNA barcode module; or (iv) a 1.sup.st DNA barcode module,
a 1.sup.st annealing site, a 2.sup.nd DNA barcode module, a
2.sup.nd annealing site, a 3.sup.rd DNA barcode module, a 3.sup.rd
annealing site, a 4.sup.th DNA barcode module, a 4.sup.th annealing
site, and a 5.sup.th DNA barcode module; or (v) a 1.sup.st DNA
barcode module, a 1.sup.st annealing site, a 2.sup.nd DNA barcode
module, a 2.sup.nd annealing site, a 3.sup.rd DNA barcode module, a
3.sup.rd annealing site, a 4.sup.th DNA barcode module, a 4.sup.th
annealing site, a 5.sup.th DNA barcode module, a 5.sup.th annealing
site, and a 6.sup.th DNA barcode module.
[0022] Moreover, what is contemplated is the above system, further
comprising a primer binding site capable of binding a DNA
sequencing primer, wherein said primer binding site is capable of
directing sequencing of one or more of the 1.sup.st DNA barcode
module, the 2.sup.nd DNA barcode module, the 3.sup.rd DNA barcode
module, the 4.sup.th DNA barcode module, the 5.sup.th DNA barcode
module, or the 6.sup.th DNA barcode module, and wherein the primer
binding site is situated 3-prime to the 1.sup.st DNA barcode
module, 3-prime to the 2nd DNA barcode module, 3-prime to the
3.sup.rd DNA barcode module, 3-prime to the 4.sup.th DNA barcode
module, 3-prime to the 5.sup.th DNA barcode module, or 3-prime to
the 6.sup.th DNA barcode module, or wherein the primer binding site
is situated in between the 1.sup.st and 2.sup.nd DNA barcode
modules, or is situated in between the 2.sup.nd and 3.sup.rd DNA
barcode modules, or is situated in between the 3.sup.rd and
4.sup.th DNA barcode modules, or is situated between the 4.sup.th
and 5.sup.th DNA barcode modules, or is situated between the
5.sup.th and 6.sup.th DNA barcode modules.
[0023] Additionally, what is provided is the above system, wherein
the primer binding site is situated in between the 1.sup.st and
2.sup.nd DNA barcode modules, or is situated in between the
2.sup.nd and 3.sup.rd DNA barcode modules, or is situated in
between the 3.sup.rd and 4.sup.th DNA barcode modules, or is
situated between the 4.sup.th and 5.sup.th DNA barcode modules, or
is situated between the 5.sup.th and 6.sup.th DNA barcode modules.
In embodiments relating to the position of a primer binding site,
relative to upstream DNA barcode modules and relative to downstream
DNA barcode modules, what is provided is the above system, wherein
a primer binding site is situated in between each and every pair of
successive DNA barcode modules.
[0024] Furthermore, what is provided is the above system, wherein
the bead comprises a DNA barcode that is an orthogonal DNA barcode,
wherein the bead comprises an external surface, and wherein the
orthogonal DNA barcode comprises: (a) A first nucleic acid that
comprises a first DNA barcode module and an annealing site for a
sequencing primer, wherein the first nucleic acid is coupled to the
bead at a first position, (b) A second nucleic acid that comprises
a second DNA barcode module and an annealing site for a sequencing
primer, wherein the second nucleic acid is coupled to the bead at a
second position, and (c) A third nucleic acid that comprises a
third DNA barcode module and an annealing site for a sequencing
primer, wherein the second nucleic acid is coupled to the bead at a
third position, and wherein the first, second, and third position
on the bead are each located at different location on the bead's
external surface.
[0025] In encoding embodiments, what is provided is the above
system, wherein the DNA barcode comprises one or more nucleic acids
that do not identify any chemical library monomer but that instead
identify: (a) The class of chemical compounds that is cleavably
attached to the bead; (b) The step number in a multi-step pathway
of organic synthesis; (c) The date that the bead-bound compound was
synthesized; (d) The disease that the bead-bound compound is
intended to treat; (e) The cellular event that the bead-bound
compound is intended to stimulate or inhibit; or (f) The reaction
conditions that were used to couple a given chemical library
monomer to the bead.
[0026] In linker embodiments, what is provided is the above system,
wherein each of the plurality of substantially identical bead-bound
compounds is coupled to the bead by way of a cleavable linker. Also
provided is the above system, wherein each of the plurality of
substantially identical bead-bound compounds is coupled to the bead
by way of a light-cleavable linker. Also provided is the above
system, wherein each of the plurality of substantially identical
bead-bound compounds is coupled to the bead by way of a
non-cleavable linker.
[0027] In TentaGel.RTM. embodiments, what is provided is the above
system, wherein the at least one bead comprises grafted copolymers
consisting of a low crosslinked polystyrene matrix on which
polyethylene glycol (PEG) is grafted.
[0028] In release-monitor embodiments, the present disclosure
provides the above system, wherein at least one picowell contains a
release-monitor bead, and does not contain any other type of
bead,
[0029] wherein the release-monitor bead comprises a bead-bound
quencher and a bead-bound fluorophore, wherein the bead-bound
quencher is quenchingly positioned in the immediate vicinity of the
bead-bound fluorophore and capable of quenching at least 50% (or at
least 60%, or at least 70%, or at least 80%, or at least 90%, or at
least 95%, or at least 99%, or at least 99.5%, or at least 99.9%)
of the fluorescence of the bead-bound fluorophore, and wherein the
bead-bound fluorophore is bound by way of a first light-cleavable
linker, wherein the picowell containing the release-monitor bead is
a first picowell, wherein the first picowell contains a first
solution, wherein exposing the first picowell to cleaving
conditions is capable of severing the light-cleavable linker and
releasing the fluorophore into the first solution of the first
picowell, wherein the exposing results in the fluorophore diffusing
throughout the first solution in the first picowell, and wherein a
fluorescent signal acquired by shining light on the first picowell
that contains the first solution comprising diffused fluorophore
allows the user to use the fluorescent signal to calculate the
percent release of the bead-bound fluorophore from the
release-monitor bead resulting in a value for the calculated
percent release, and wherein a second picowell contains a
bead-bound compound coupled with the same type of light-cleavable
linker as the first light-cleavable linker, and wherein the second
picowell contains a second solution,
[0030] and wherein the value for the calculated percent release
from the release-monitor bead in the first picowell allows
calculation of the concentration of the released compound in the
second solution of the second picowell.
[0031] In embodiments relating to identity of all of the compounds
bound to a given bead, or relating to identity of all of the DNA
barcodes bound to a given bead, what is provided is the above
system, wherein the at least one bead comprises a plurality of
substantially identical bead-bound DNA barcodes, wherein the
plurality is between 10 million to 100 million copies of the
substantially identical bead-bound DNA barcodes. Also provided is
the above system, wherein the at least one bead comprises a
plurality of substantially identical bead-bound compounds, where
wherein the plurality is between 10 million to 100 million copies
of the substantially identical bead-bound compounds.
[0032] In embodiments relating to cells (e.g., mammalian cells,
cancer cells, bacterial cells), what is provided is the above
system, wherein at least one picowell comprises at least one cell,
wherein the plurality of substantially identical bead-bound
compounds are bound to the at least one bead by way of a cleavable
linker, and wherein cleaving the cleavable linker releases the
bead-bound compound from the bead to produce a released compound,
and wherein the released compound is capable of contacting the at
least one cell. In other cell embodiments, what is provided is the
above system, wherein at least one picowell comprises at least one
cell, wherein the plurality of substantially identical bead-bound
compounds are bound to the at least one bead by way of a cleavable
linker, and wherein cleaving the cleavable linker releases the
bead-bound compound from the bead to produce a released compound,
and wherein the released compound is capable of contacting the at
least one cell, and wherein the at least one cell is: (i) a
mammalian cell that is not a cancer cell, (ii) a mammalian cancer
cell, (iii) a dead mammalian cell, (iv) an apoptotic mammalian
cell, (v) a necrotic mammalian cell, (vi) a bacterial cell, (vii) a
plasmodium cell, (vii) a cell that is metabolically active but has
a cross-linked genome and is unable to undergo cell division, or
(ix) a mammalian cell that is infected with a virus.
[0033] In device embodiments, what is provided is the above system,
wherein each picowell has a top aperture that defines an opening at
the top of the picowell, a bottom that is defined by a floor,
wherein the top aperture is separated from the floor, and wherein a
wall resides in between the top aperture and the floor, and wherein
the aperture is round, wherein the floor is round, and wherein the
wall takes the form of a truncated cone, and wherein the aperture
has a first diameter, the floor has a second diameter, and wherein
the first diameter is greater than the second diameter.
[0034] In other device-related embodiments, what is provided is the
above system, wherein each picowell has a top aperture that defines
an opening at the top of the picowell, a bottom that is defined by
a floor, wherein the top aperture is separated from the floor, and
wherein a wall resides in between the top aperture and the floor,
and wherein the aperture is round, wherein the floor is round, and
wherein the wall takes the form of a truncated cone, and wherein
the aperture has a first diameter, the floor has a second diameter,
and wherein the first diameter is greater than the second diameter,
further comprising a cap that snuggly fits into the aperture,
wherein the aperture is comprised by a polymer having a greater
durometer (harder) and wherein the cap is made of a polymer having
a lesser durometer (softer), and wherein the relative durometers of
the cap and aperture allow the cap to be reversibly and snuggly fit
into the aperture, and wherein the cap is: (i) a cap intended only
to plug the picowell and prevent leakage, (ii) a cap that is a
passive cap and that is capable of absorbing metabolites that are
released by a cell, in the situation where a cell in a cell medium
is cultured in the picowell, (iii) a cap that is an active cap, and
that takes the form of a bead that comprises a plurality of
essentially identical compounds, and wherein each of the plurality
of essentially identical compounds is coupled to the bead with a
cleavable linker; (iv) a cap that is an active cap, and that takes
the form of a bead that comprises a plurality of identical
reagents, and wherein each of the plurality of essentially
identical reagents is coupled to the bead with a cleavable linker.
Also provided is the above system, wherein the cap is spherical, or
wherein the cap is non-spherical.
[0035] In embodiments, the above system comprises a picowell array
plate comprising an upper generally planar surface, a plurality of
picowells, wherein each picowell has a top aperture that defines an
opening at the top of the picowell, a bottom that is defined by a
floor, wherein the top aperture is separated by a wall from the
floor, and wherein the wall resides in between the top aperture and
the floor, and optionally, a bead disposed in at least one of said
plurality of picowells, wherein the bead comprises a plurality of
substantially identical bead-bound DNA barcodes, and a plurality of
substantially identical bead-bound compounds, wherein the picowell
array plate further comprises a mat that is capable of securely
covering the opening at the top of at least one or all of the
plurality of picowells, or that is actually securely covering the
opening at the top of at least one or all of the plurality of
picowells, wherein the securely covering is reversible, wherein the
mat optionally comprises one or all of: (a) An absorbant surface
that, when positioned in contact with the upper generally planar
surface of the picowell array plate, is capable of absorbing any
metabolites, biochemicals, or proteins that may be comprised by one
or more of the plurality of picowells, (b) An adhesive surface that
is capable of maintaining reversible adhesion to the top generally
planar surface of the picowell array plate.
[0036] In biochemical assay embodiments, what is embraced is the
above system, that includes at least one picowell, wherein the at
least one picowell comprises a bead that comprises a plurality of
substantially identical compounds and a plurality of substantially
identical barcodes, wherein the at least one picowell comprises an
assay medium that includes cereblon E3 ubiquitin ligase, a
substrate of cereblon E3 ubiquitin ligase such as Ikaros or Aiolos,
and wherein the system is capable of screening for compounds that
activate cereblon's E3 ubiquitin ligase activity, and are thereby
capable of reducing intracellular concentrations of Ikaros or
Aiolos.
[0037] In another biochemical assay embodiment, what is
contemplated is the above system, that includes at least one
picowell, wherein the at least one picowell comprises a bead that
comprises a plurality of substantially identical compounds and a
plurality of substantially identical barcodes, wherein the at least
one picowell comprises an assay medium that includes MDM2 E3
ubiquitin ligase, a substrate of MDM2 E3 ubiquitin ligase such as
p53, and wherein the system is capable of screening for compounds
that activate MDM2's E3 ubiquitin ligase activity, and thereby
capable of increasing the intracellular concentrations of p53.
[0038] In more barcoding embodiments, what is provided is the above
system, wherein the DNA barcode comprises one or more nucleic acids
that do not encode any chemical monomer but that instead identify
one or more of: (a) The class of chemical compounds that is
cleavably attached to the bead; (b) The step in a multi-step
pathway of organic synthesis, wherein a bead-bound nucleic acid
corresponds to a given chemical monomer that is used to make a
bead-bound compound, and wherein the bead-bound nucleic acid that
corresponds to a given chemical monomer identifies that chemical
monomer; (c) The date that the bead-bound compound was synthesized;
(d) The disease that the bead-bound compound is intended to treat;
(e) The cellular event that the bead-bound compound is intended to
stimulate or inhibit.
[0039] In embodiments that lack any headpiece, what is provided is
the above system, wherein the at least one bead comprises a
plurality of substantially identical bead-bound compounds and also
comprises a plurality of substantially identical bead-bound DNA
barcodes, and wherein there does not exist any headpiece that links
any of the bead-bound compounds to any of the bead-bound DNA
barcodes.
[0040] Moreover, what is contemplated is the above system, wherein
at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% of the substantially identical bead-bound DNA barcodes have an
identical structure. Additionally, what is contemplated is the
above system, wherein at least 70%, at least 80%, at least 90%, at
least 95%, or at least 98% of the substantially identical
bead-bound compounds have an identical structure.
[0041] Furthermore, what is supplied is the above system, wherein
the concatenated DNA barcode comprises at least one nucleic acid
that is a DNA barcode module, or the above system, wherein the
concatenated DNA barcode comprises only one nucleic acid that is a
DNA barcode module.
[0042] In sequencing primer annealing site embodiments, what is
provided is the above system, wherein the concatenated DNA barcode
comprises at least one nucleic acid that is a DNA barcode module,
and at least one functional nucleic acid that: (a) Is capable of
being used as an annealing site for a sequencing primer, (b) Is
capable of forming a hairpin structure, and wherein the hairpin
structure comprises a sequencing primer, an annealing site for the
sequencing primer, and a bend in the hairpin structure wherein the
bend is 5-prime to the sequencing primer and is 3-prime to the
annealing site for the sequencing primer, or (c) Is a spacer
nucleic acid.
[0043] In other sequencing primer embodiments, what is provided is
the above system, wherein the orthogonal DNA barcode contains a
plurality of DNA barcode modules, wherein each of the DNA barcode
modules is coupled to a different site on the bead either directly
or via a linker, and wherein each of the plurality of DNA barcode
modules contains at least one functional nucleic acid that: (a) Is
capable of being used as an annealing site for a sequencing primer,
(b) Is capable of forming a hairpin structure, and wherein the
hairpin structure comprises a sequencing primer, an annealing site
for the sequencing primer, and a bend in the hairpin structure
wherein the bend is 5-prime to the sequencing primer and is 3-prime
to the annealing site for the sequencing primer, or (c) Is a spacer
nucleic acid.
[0044] In embodiments that recite functional language about splint
oligos, what is provided is a bead comprising a concatenated DNA
barcode, wherein the concatenated DNA barcode comprises: (a) a
first DNA barcode module and a first annealing site for a first
splint oligonucleotide (splint oligo), wherein the splint oligo
comprises three nucleic acids, wherein the three nucleic acids are:
a nucleic acid that is a hybridizing complement to the first
annealing site, a nucleic acid that is a hybridizing complement to
a 2.sup.nd DNA barcode module, and a nucleic acid that is a
2.sup.nd annealing site, and (b) a second DNA barcode module and a
2nd annealing site for a second splint oligo, wherein the second
splint oligo comprises three nucleic acids, wherein the three
nucleic acids are: a nucleic acid that is a hybridizing complement
to the 2nd annealing site, a nucleic acid that is a 3rd DNA barcode
module, and a nucleic acid that is a 3rd annealing site.
[0045] In another embodiment that contains functional language
relating to splint oligos, what is provided is the above bead,
further comprising: a third DNA barcode module and a 3rd annealing
site for a third splint oligo, wherein the third splint oligo
comprises three nucleic acids, wherein the three nucleic acids are:
a nucleic acid that is a hybridizing complement to the 3rd
annealing site, a nucleic acid that is a 4.sup.th DNA barcode
module, and a nucleic acid that is a 4th annealing site.
[0046] Moreover, in yet another embodiment containing functional
language relating to splint oligos, what is provided is the above
bead, further comprising one or more of: (i) a fourth DNA barcode
module and a 4th annealing site for a fourth splint oligo, wherein
the fourth splint oligo comprises three nucleic acids, wherein the
three nucleic acids are: a nucleic acid that is a hybridizing
complement to the 4th annealing site, a nucleic acid that is a
5.sup.th DNA barcode module, and a nucleic acid that is a 5th
annealing site, (ii) a response capture element, (iii) a release
monitor.
[0047] In linker embodiments, what is embraced is the above bead,
wherein the concatenated DNA barcode is coupled to the bead, but
is: (i) not coupled to the bead by way of any photocleavable
linker, (ii) not coupled to the bead by any enzymatically cleavable
linker; or (iii) not coupled to the bead by any kind of cleavable
linker.
[0048] In an embodiment relating to distinct coupling positions,
what is provided is the above bead, wherein the concatenated DNA
barcode is coupled to a first position on the bead, wherein the
bead also comprises a compound that is coupled to a second position
on the bead, and wherein the first position is not the same as the
second position.
[0049] In surface embodiments (interior and exterior surfaces),
what is provided is the above bead, wherein the bead comprises an
exterior surface and an interior surface, wherein the bead
comprises at least 10,000 substantially identical concatenated DNA
barcodes that are coupled to the bead, and wherein at least 90% of
the at least 10,000 substantially identical concatenated DNA
barcodes are coupled to the exterior surface.
[0050] In exclusionary embodiments that can distinguish the present
disclosure from other embodiments, what is provided is the above
bead, that is does not comprise any polyacrylamide, and wherein the
concatenated DNA barcode: (i) Does not include any nucleic acid
that is a promoter; (ii) Does not include any nucleic acid that is
polyA; or (iii) Does not include any nucleic acid that is a
promoter and does not include any nucleic acid that is polyA.
[0051] In release-monitor bead embodiments, the present disclosure
supplies a release-monitor bead that is capable of functioning in
an aqueous medium, wherein the release-monitor bead comprises a
bead-bound quencher and a bead-bound fluorophore, wherein the
bead-bound quencher is quenchingly positioned in the immediate
vicinity of the bead-bound fluorophore and capable of quenching at
least 50% of the fluorescence of the bead-bound fluorophore, and
wherein the bead-bound fluorophore is bound by way of a first
light-cleavable linker, wherein the picowell containing the
release-monitor bead is a first picowell, wherein the first
picowell contains a first solution, wherein exposing the first
picowell to cleaving conditions is capable of severing the
light-cleavable linker and releasing the fluorophore into the first
solution of the first picowell, wherein the exposing results in the
fluorophore diffusing throughout the first solution in the first
picowell, and wherein a fluorescent signal acquired by shining
light on the first picowell that contains the first solution
comprising diffused fluorophore allows the user to use the
fluorescent signal to calculate the percent release of the
bead-bound fluorophore from the release-monitor bead resulting in a
value for the calculated percent release, and wherein a second
picowell contains a bead-bound compound coupled with the same type
of light-cleavable linker as the first light-cleavable linker, and
wherein the second picowell contains a second solution, and wherein
the value for the calculated percent release from the
release-monitor bead in the first picowell allows calculation of
the concentration of the released compound in the second solution
of the second picowell. In other release-monitor embodiments, what
is provided is a release-monitor bead wherein the fluorophore is
TAMRA and wherein the quencher is QSY7, and a release-monitor bead
that has the structure shown in FIG. 9, and a release-monitor bead
of that has the structure shown in FIG. 10, and a release-monitor
bead, wherein the capable of quenching is at least 90%, at least
98%, at least 99%, or at least 99.9%.
[0052] In a methods of manufacture embodiment, what is embraced is
a method for synthesizing a release-monitor bead, wherein the
release-monitor bead comprises a bead, a quencher, a fluorophore,
and a photocleavable linker that couples the fluorophore to the
bead, the method comprising, in this order, (i) Providing a resin,
(ii) Coupling a lysine linker to the resin, wherein the reagent
containing the lysine linker is L-Fmoc-Lys(4-methyltrityl)-OH,
(iii) Removing the Fmoc protecting group, (iv) Coupling the
quencher using a reagent that is quencher-N-hydroxysuccinimide
(quencher-NETS) as the source of quencher, (v) Removing the
4-methyltrityl protecting group using a reagent comprising
trifluoroacetic acid, (vi) Coupling a photocleavable linker to the
epsilon amino group of lysine, wherein the photocleavable linker is
provided by a reagent that is, Fmoc-photocleavable linker-OH, (vii)
Coupling the fluorophore. Also provided is the above embodiment,
but without regard to the ordering of steps. In other methods
embodiments, what is provided is the above method wherein the
fluorophore is TAMRA and wherein the quencher is QSY7.
[0053] In methods relating to the utility of release-monitor bead,
what is provided is a method for controlling the concentration of a
compound in a solution that resides in a picowell, wherein the
method is applied to a bead-bound compound in a picowell, wherein
the picowell contains a solution, and wherein the bead-bound
compound is coupled to the bead by way of a cleavable linker, the
method comprising: (a) Exposing the bead-bound compound to a
condition that effects cleavage of the cleavable linker and
releases the bead-bound compound from the bead to generate a
released compound, wherein release is followed by diffusion or
dispersion of the released compound in the solution to result in a
substantially uniform concentration of the compound in the
solution, (b) Wherein the condition comprises light that is capable
of cleaving the cleavable linker, (c) Wherein the condition is
adjusted to produce a determined concentration of the substantially
uniform concentration, and (d) Wherein the determined concentration
is made with regard to the concentration of a released fluorophore
that is released by from a bead-bound release-monitor. Provided
also, is the above method, wherein the condition is adjusted by
adjusting one or more of the wavelength of the light, the intensity
of the light, and by the duration of light exposure, and the above
method, wherein the concentration of a released fluorophore that is
released from a bead-bound release-monitor is determined at the
same time as effecting release of the bead-bound compound from the
bead to generate a released compound, and the above method, wherein
the concentration of a released fluorophore that is released from a
bead-bound release-monitor is determined at a time substantially
before effecting release of the bead-bound compound from the bead
to generate a released compound.
[0054] The term "determined" can mean a concentration that is
predetermined and decided upon as being a desired concentration,
prior to exposing the bead to light. Also, the term "determined"
can mean a concentration that is decided upon in "real time," that
is, a concentration that is decided upon at the same time as the
exposing the bead to light.
[0055] In cap embodiments, what is embraced is a cap in combination
with a picowell plate that comprises a plurality of picowells,
wherein the cap is capable of use with the picowell plate that
comprises a plurality of picowells, wherein each of the plurality
of picowells is definable by an aperture, a floor, and a wall,
wherein the wall is defined by the aperture on top and the floor on
the bottom, and wherein the aperture is round, wherein the floor is
round, and wherein the wall takes the form of a surface of a
truncated cone, and wherein the aperture has a first diameter, the
floor has a second diameter, and wherein the first diameter is
greater than the second diameter, wherein the cap is a spherical
cap that is capable of snuggly fitting into the aperture, wherein
the aperture is comprised by a polymer having a greater durometer
(harder) and wherein the cap is made of a polymer having a lesser
durometer (softer), and wherein the relative durometers of the cap
and aperture allow the spherical cap to be reversibly and snuggly
fit into the aperture, and wherein the cap is: (i) capable of
plugging the picowell and preventing leakage, (ii) a passive cap
and that is capable of absorbing metabolites that are released by a
cell, in the situation where a cell in a cell medium is cultured in
the picowell, (iii) an active cap that takes the form of a bead
that comprises a plurality of essentially identical compounds, and
wherein each of the plurality of essentially identical compounds is
coupled to the bead with a cleavable linker, and wherein cleavage
of the cleavable linker releases at least some of the plurality of
compounds from the bead, (iv) an active cap that takes the form of
a bead that comprises a plurality of identical reagents, and
wherein each of the plurality of essentially identical reagents is
coupled to the bead with a cleavable linker, and wherein cleavage
of the cleavable linker releases at least some of the plurality of
reagents from the bead.
[0056] In porous cap embodiments, what is provided is a plurality
of porous caps in combination with a picowell plate and a solid
polymer coating, wherein each of the plurality of porous caps
comprises an upper surface and a lower surface, wherein the
picowell plate comprises a plurality of picowells, wherein at least
one porous cap contacts a picowell and reversibly and snuggly fits
into the picowell, wherein the picowell plate and each of the upper
surfaces of the plurality of porous caps is covered with a solid
polymer coating, wherein the solid polymer coating contacts at
least some of the upper surface of each cap and is adhesively
attached to said at least some of the upper surface, and wherein,
(i) Each of the plurality of picowells is capable of holding an
aqueous solution, wherein products of a reaction are generated in
the solution, and wherein at least some of the products are
absorbed by the lower surface of each of the plurality of porous
caps, (ii) Wherein a solution of a polymerizable reagent that
capable of polymerization is poured over the plurality of porous
caps in combination with the picowell plate, and wherein the
polymerizable reagent is polymerized to form a substantially planar
surface that coats substantially all of the top surface of the
picowell plate, thereby fixing the polymerized reagent to each of
the plurality of porous caps, and (iii) Wherein all of the
plurality of porous caps are removable by the act of peeling from
the plurality of picowells, wherein adhesion is maintained between
the plurality of porous caps and the polymerized reagent, resulting
in an array of adhering caps partly with the upper surface of each
cap is embedded in the polymerized reagent and the lower surface of
each cap is accessible for analysis of any absorbed reaction
product.
[0057] This provides a methods of manufacture embodiment, for using
splint oligos to guide the enzymatic synthesis of a DNA barcode.
What is provided is a method for making a bead-bound concatenated
DNA barcode, wherein the bead-bound concatenated DNA barcode
comprises a plurality of DNA barcode modules, and optionally one or
more functional nucleic acids, and optionally one or more
identity-encoding nucleic acids that encode the identity of
something other than the identity of a chemical library monomer,
the method comprising: (a) The step of providing a bead with a
coupled polynucleotide that comprises a 1.sup.st DNA barcode module
and a 1.sup.st annealing site, wherein the 1.sup.st annealing site
is capable of hybridizing with a first splint oligonucleotide
(splint oligo), the first splint oligo being capable of serving as
a template for DNA polymerase to catalyze the polymerization to the
coupled polynucleotide, nucleotides that are complementary to those
of the hybridized first splint oligo, wherein the polymerized
nucleotides that are complementary to those of the hybridized first
splint oligo following polymerization comprise a bead-bound
2.sup.nd DNA barcode module and a 2.sup.nd annealing site; (b) The
step of providing said bead with a coupled polynucleotide with said
first splint oligo, and allowing said first splint oligo to
hybridize with said coupled polynucleotide; (c) The step of adding
a DNA polymerase and deoxynucleotide triphosphates (dNTPs) and
allowing the DNA polymerase to catalyze polymerization of said
dNTPs to the coupled polynucleotide, wherein the coupled
polynucleotide has a free 3'-terminus and wherein the
polymerization is to the free 3'-terminus, (d) The step of washing
away the first splint oligo. Also contemplated is the above method,
wherein the first splint oligo comprises a 1.sup.st annealing site,
a 2.sup.nd DNA barcode module, and a 2.sup.nd annealing site.
[0058] In further methods of manufacture embodiments, what is
provided is the above method, wherein the first splint oligo
comprises a 1.sup.st annealing site, a 2.sup.nd DNA barcode module,
a 2.sup.nd annealing site, and a nucleic acid encoding a 1.sup.st
sequencing primer annealing site, wherein the 1.sup.st sequencing
primer annealing site is capable of hybridizing to a sequencing
primer resulting in a hybridized sequencing primer, and wherein the
hybridized sequencing primer is capable of directing the sequencing
of the 2.sup.nd DNA barcode module and the 1.sup.st DNA barcode
module.
[0059] Moreover, what is contemplated is the above method, wherein
the first splint oligo, the DNA polymerase, and the dNTPs are all
added at the same time, or wherein the first splint oligo, the DNA
polymerase, and the dNTPs are each added at separate times.
[0060] Regarding interior versus exterior locations on a bead, what
is provided is the above method, wherein the bead comprises an
exterior location and an interior location, and wherein the
bead-bound concatenated DNA barcode is coupled to the bead at
locations that are substantially on the exterior of the bead and
sparingly at interior locations of the bead, and wherein the bead
also comprises a plurality of coupled compounds wherein all of the
plurality of coupled compounds have substantially an identical
structure, when compared to each other, and wherein the bead is
comprised substantially of a hydrophobic polymer.
[0061] In further methods embodiments, what is provided is the
above method, further comprising: (a) The step of providing a bead
with a coupled first longer polynucleotide that comprises a
1.sup.st DNA barcode module, a 1.sup.st annealing site, a 2.sup.nd
DNA barcode, and a 2.sup.nd annealing site, wherein the 2.sup.nd
annealing site is capable of hybridizing with a second splint
oligo, the second splint oligo being capable of serving as a
template for DNA polymerase to catalyze the polymeraztion to the
coupled first longer polynucleotide, nucleotides that are
complementary to those of the hybridized second splint oligo,
wherein the polymerized nucleotides that are complementary to those
of the hybridized second splint oligo following polymerization
comprise a bead-bound 3.sup.rd DNA barcode module and a 3.sup.rd
annealing site; (b) The step of providing said bead with a coupled
polynucleotide with said 2.sup.nd splint oligo, and allowing said
2' splint oligo to hybridize with said coupled first longer
polynucleotide; (c) The step of adding a DNA polymerase and
deoxynucleotide triphosphates (dNTPs) and allowing DNA polymerase
to catalyze polymerization of said dNTPs to the coupled longer
polynucleotide, wherein the coupled longer polynucleotide has a
free 3'-terminus and wherein the polymerization is to the free
3'-terminus, (d) The step of washing away the second splint
oligo.
[0062] This relates to the consecutive numbering of the first DNA
barcode module, the second DNA barcode module, the third DNA
barcode module, and so on, for the manufacture of the entire DNA
barcode. This also relates to repeating the cycle of methods steps,
over and over and over, in the manufacture of the entire DNA
barcode. What is provided is the above method, wherein each of said
plurality of DNA barcode modules is identified or named by a
number, the method further comprising reiterating the recited
steps, where for a first reiteration, the name of the DNA barcode
module is increased by adding one number to the existing name, the
name of the annealing site is increased by adding one number to the
existing name, and the name of the splint oligo is increased by
adding one number to the name o the existing distal terminal DNA
barcode module, and the name of the "first longer polynucleotide"
is changed by adding one number to the existing name, wherein the
comprising reiterating the recited steps is one reiteration, or two
reiterations, or three reiterations, or four reiterations, or five
reiterations, or more than five reiterations, or more than ten
reiterations.
[0063] Also contemplated is the above method, that comprises a
plurality of splint oligos, wherein each splint oligo comprises a
sequencing primer annealing site, wherein the sequencing primer
annealing site is capable of hybridizing to a sequencing primer
resulting in a hybridized sequencing primer, and wherein the
hybridized sequencing primer is capable of directing the sequencing
of the at least one bead-bound DNA barcode module and at least one
bead-bound DNA barcode module.
[0064] This concerns embodiments relating to splint oligos that
guides DNA polymerase to synthesize functional nucleic acids and
various types of informative nucleic acids. What is provided is the
above method, wherein at least one splint oligo comprises a
functional nucleic acid, or wherein at least one splint oligo
encodes information other than information on a chemical library
monomer. What is provided is the above method, further comprising
the step of coupling of at least one DNA barcode module by way of
click chemistry, wherein the step does not use any splint
oligo.
[0065] Briefly stated, the present disclosure provides a system for
screening chemical compounds, comprising: (a) A picowell array
plate comprising a plurality of picowells, wherein each picowell
has a top aperture that defines an opening at the top of the
picowell, a bottom that is defined by a floor, wherein the top
aperture is separated from the floor, and wherein a wall resides in
between the top aperture and the floor; (b) At least one bead
disposed in at least one picowell, wherein the at least one bead
comprises a plurality of substantially identical bead-bound DNA
barcodes, and a plurality of substantially identical bead-bound
compounds, (c) Wherein the at least one bead comprises a DNA
barcode that takes the form of either a concatenated DNA barcode or
an orthogonal DNA barcode, and wherein if the DNA barcode takes the
form of a concatenated DNA barcode the concatenated DNA barcode is
made using a method that: (i) Uses click chemistry, or (ii) Uses a
repeating cycle of steps, wherein the steps in the repeating cycle
comprise using a splint oligo for annealing to a partially made DNA
barcode, wherein the annealed splint oligo is used as a template
for extending the partially made DNA barcode using DNA polymerase,
and wherein the splint oligo contains bases that are complementary
to a DNA barcode module that is to be polymerized to the partially
made DNA barcode.
[0066] In another aspect, what is provided is the above system,
wherein the DNA barcode comprises: (a) One or more DNA barcode
modules wherein each of the one or more DNA barcode modules encodes
information on the identity of a chemical library monomer, and (b)
Optionally one or more functional nucleic acids, and (c)
Optionally, one or more nucleic acids that encode information that
a type of information other than information on the identity of a
chemical library monomer.
[0067] Moreover, what is provides is the above system, further
comprising a plurality of caps, each capable of fitting into the
opening of a different picowell, and each capable of minimizing or
preventing evaporation of fluid that is inside of the picowell, and
each capable of minimizing or preventing leakage of fluid that is
inside of the picowell.
[0068] Also embraced is the above system, further comprising a
plurality of spherical caps, wherein each is capable of fitting
into the aperture of a picowell wherein the aperture is circular,
and each capable of minimizing or preventing evaporation of fluid
that is inside of the picowell, and each capable of minimizing or
preventing leakage of fluid that is inside of the picowell.
[0069] Also contemplated is the above system, wherein if the at
least one bead comprises a DNA barcode that takes the form of a
concatenated DNA barcode, the concatenated DNA barcode comprises:
(i) A sequencing primer binding site, (ii) A first DNA barcode
module, (iii) A first annealing site that is capable of hybridizing
with a first oligonucleotide splint, wherein the first
oligonucleotide splint is capable of being used to guide the
enzymatic synthesis of a second DNA barcode module, (iv) A second
DNA barcode module, (v) A second annealing site that is capable of
hybridizing with a second oligonucleotide splint, wherein the
second oligonucleotide splint is capable of being used to guide the
synthesis of a third DNA barcode, (vi) A third DNA barcode module,
(vii) A third annealing site that is capable of hybridizing with a
third oligonucleotide splint, wherein the third oligonucleotide
splint is capable of being used to synthesize a fourth DNA
barcode.
[0070] In methods embodiments, what is provided is a method for
screening a compound library for compounds having desired
properties, comprising: (a) providing a plurality of beads, wherein
each bead comprises a plurality of oligonucleotides attached to the
bead surface and a plurality of substantially related compounds
attached to the bead surface, and wherein the sequence of the
oligonucleotides attached to the beads encodes the synthesis
history of the plurality of substantially related compounds
attached to the bead surface; (b) incorporating the plurality of
beads in an assay for desired properties of compounds in the
compound library; (c) capturing a signal from at least one bead,
wherein the signal reflects the performance of the compounds on the
bead in the assay; (d) sequencing the plurality of oligonucleotides
attached to the at least one bead for which assay signal was also
captured, without removing the oligonucleotides from the bead; and
(e) identifying at least one compound from the sequencing readout
of step (d) and relating it to its corresponding assay performance
captured in the signal of step (c).
[0071] In further detail, what is embraced is the above method,
wherein the assay comprises a binding assay, or wherein the assay
comprises an activity assay, or wherein the assay comprises a
competitive binding assay or a competitive inhibition assay, or
wherein the assay comprises interaction of untethered compounds
with other assay reagents, wherein the untethered compounds are
compounds released from the bead surface, or wherein the compounds
are released by cleaving a cleavable linker that connects the
compounds to the beads, or wherein the assay occurs in a plurality
of confined volumes, wherein nominally one bead is dispersed per
confined volume.
[0072] In another aspect, what is further contemplated, is the
above method, wherein the confined volume comprises an aqueous
droplet, or
[0073] wherein the aqueous droplet is suspended in an oil medium or
a hydrophobic liquid medium, or wherein the confined volume
comprises a picowell, or wherein the picowells are organized in a
regular array, or wherein the plurality of confined volumes are
organized in a regular array.
[0074] Moreover, what is further embraced is the above method,
wherein the confined volume comprises a layer of adherent aqueous
medium around the bead, wherein the bead is suspended in a
hydrophobic medium, and the above method, wherein the assay
reagents are washed away before sequencing the oligonucleotides.
And the above method wherein the sequencing step (d) is performed
before the assay step (b). What is also provided is the above
method, wherein the oligonucleotides on the beads are removed after
the sequencing step, but before the assay step. Moreover, further
contemplated is the above method, wherein the removing of the
oligonucleotide comprises an enzymatic digestion, a chemical
cleavage, a thermal degradation or a physical shearing, and the
above method, wherein the binding assay comprises binding of RNA
molecules to the beads, and the above method, wherein the signal
from the bead comprises sequencing of the bound RNA molecules.
[0075] In yet another aspect, what is provided is the above method,
wherein the binding assay comprises a fluorescently labeled binding
assay, wherein the molecules binding to the compounds on the beads
comprise fluorophores, or the above method, wherein the binding
assay comprises nucleic-acid labeled binding assay, wherein the
molecules binding to the compounds on the beads comprise
nucleic-acid tags, wherein further the signal from the assay
comprises sequencing of the nucleic acid tags attached to the
molecules binding to the compounds on the beads.
[0076] In yet a methods embodiment relating to properties, what is
provided is the above method, wherein the desired properties
include one or more of: (i) Inhibiting or stimulating the catalytic
activity of an enzyme, (ii) Stimulating Th1-type immune response,
as measurable by cell-based assays or by in vivo assays, (iii)
Stimulating Th2-type immune response, as measurable by cell-based
assays or by in vivo assays, (iv) Inhibiting Th1-type immune
response, as measurable by cell-based assays or by in vivo assays,
(v) Inhibiting Th2-type immune response, as measurable by
cell-based assays or by in vivo assays, (vi) Stimulating or
inhibiting ubiquitin-mediated degradation of a protein, as
measurable by purified proteins, by cell-based assay, or by in vivo
assays.
[0077] In a system embodiment, what is provided is a system for
screening a compound library for a compound having a desired
activity, comprising: (a) a sample compartment for receiving a
plurality of compound-attached, oligonucleotide-encoded beads; (b)
a plurality of encapsulation compartments within the sample
compartment, each encapsulation compartment nominally comprising a
single bead dispersed in an assay medium, wherein further the assay
medium comprises reagents whose interaction with the compounds on
the beads is being assayed resulting in a measurable signal; (c) a
detector for measuring signals; (d) a sequencing platform; and (e)
a user interface for receiving one or more commands from a user.
Also provided is the above system, wherein the encapsulation
compartment comprises a liquid droplet. In another aspect, provided
is the above system, wherein the encapsulation compartment
comprises a picowell, or wherein further the encapsulation
compartment comprises assay reagents, or wherein the detector
comprises an optical detector, or wherein the sequencer comprises
the optical detector.
[0078] In one aspect, the disclosure features a method for
perturbing a cell by: (a) providing a nucleic-acid encoded
perturbation and confining a cell with the nucleic-acid encoded
perturbation; (b) contacting the cell with the nucleic-acid encoded
perturbation in a confined volume, wherein the perturbation
initiation and dose are controlled; (c) incubating the cell with
the nucleic-acid encoded perturbation for a specified period of
time; and (d) transferring the nucleic acid that encodes the
nucleic-acid encoded perturbation to the cell.
[0079] In some embodiments of this aspect, the nucleic-acid encoded
perturbation is a nucleic acid encoded compound or drug molecule.
In some embodiments, the nucleic-acid encoded perturbation is a
DNA-encoded library.
[0080] In some embodiments, the perturbation and the nucleic acid
encoding the perturbation are unattached and free in solution. In
some embodiments, the perturbation and the nucleic acid encoding
the perturbation are attached to each other. In some embodiments,
the perturbation and the nucleic acid encoding the perturbation are
attached to the same substrate but not to each other. In some
embodiments, the attachment of the perturbation to the substrate
and the attachment of the nucleic acid to the substrate are
cleavable attachments. In particular embodiments, the cleavable
attachment is selected from the group consisting of a
photocleavable attachment, a temperature cleavable attachment, a pH
sensitive attachment, an acid cleavable attachment, a base
cleavable attachment, a sound cleavable attachment, a salt
cleavable attachment, a redox sensitive attachment, or a physically
cleavable attachment.
[0081] In some embodiments of this aspect of the disclosure,
confining the cell and the perturbation comprises a droplet
encapsulation, an emulsion encapsulation, a picowell encapsulation,
a macrowell encapsulation, a physical attachment, a bubble
encapsulation, or a microfluidic confinement.
[0082] In some embodiments, the control over the perturbation
comprises controlling light exposure, controlling temperature
exposure, controlling pH exposure, controlling time exposure,
controlling sound exposure, controlling salt exposure, controlling
chemical or physical redox potential, or controlling
mechanical-agitation exposure.
[0083] In particular embodiments, the incubation comprises exposing
the cell to the perturbation after cleaving the perturbation from
the substrate or after cleaving the nucleic acid from the
substrate. In some embodiments, the incubation comprises exposing
the cell to the perturbation without cleaving the perturbation from
the substrate or without cleaving the nucleic acid from the
perturbation.
[0084] In some embodiments, transferring the nucleic acid that
encodes the nucleic-acid encoded perturbation to the cell comprises
attaching the nucleic acid to the cell surface of the cell. In
particular embodiments, attaching the nucleic acid to the cell
surface of the cell comprises intercalating the nucleic acid into
the cell membrane. In particular embodiments, attaching the nucleic
acid to the cell surface of the cell comprises attaching the
nucleic acid to a biomolecule on the cell surface. In particular
embodiments, the biomolecule is a protein or a carbohydrate. In
other embodiments, attaching the nucleic acid to the cell surface
of the cell comprises attaching through an optional tag on the
nucleic acid.
[0085] In another aspect, the disclosure features a method for
perturbing a cell with a perturbation and encoding the cell with
the identity of the perturbation. The method includes: (a)
providing a bead-bound DNA encoded library; (b) confining a cell
with the bead-bound DNA encoded library, wherein the bead-bound DNA
encoded library comprises one or more copies of a combinatorially
synthesized compound and one or more copies of an encoding nucleic
acid tag, wherein the compound and the encoding nucleic acid are
attached to a bead, wherein the encoding nucleic acid encodes the
identity of the compound, and wherein the bead-bound DNA encoded
library and the cell are confined in a confining volume; (c)
releasing the compound from the bead and incubating the compound
with the cell inside the confining volume; (d) optionally releasing
the encoding nucleic acid tag from the bead; and (e) attaching the
encoding nucleic acid tag to the cell, thereby preserving the
identity of the compound through the encoding nucleic acid tag
attached to the cell.
[0086] In yet another aspect, the disclosure features a method for
perturbing a cell, encoding the cell with the identity of the
perturbation, and measuring a response of the cell to the
perturbation. The method includes: (a) contacting a cell with a
bead-bound DNA encoded library in a first confined volume, wherein
the bead-bound DNA encoded library comprises one or more copies of
a combinatorially synthesized compound and one or more copies of an
encoding nucleic acid tag, wherein the compound and the encoding
nucleic acid are attached to a bead, and wherein the encoding
nucleic acid encodes the identity of the compound; (b) releasing
the compounds in the library from the bead and incubating the
compounds in the library with the cell inside the first confined
volume; (c) optionally releasing the encoding nucleic acid tag from
the bead inside the first confined volume; (d) capturing the
encoding nucleic acid tag to the cell surface of the cell, whereby
the cell is exposed to the compound in the library and the identity
of the compound exposed is captured on to the cell surface; (e)
releasing the cell from the first confining volume, wherein the
encoding nucleic acid tags are attached to the cell and the
encoding nucleic acid tag encodes the identity of the compound the
cell is exposed to; (f) capturing a previously perturbed and
nucleic acid tagged cell with a response-detection bead in a second
confined volume, wherein the cell is exposed to a lysis condition
that exposes the cellular content of the cell to the
response-capture bead, wherein the response-capture bead comprises
capture probes that capture the cellular content and the nucleic
acid tag that encodes the perturbation in the previously perturbed
and nucleic acid tagged cell; (g) incubating the response-capture
bead with the lysed cell in the second confining volume, thereby
capturing both cellular content and the nucleic acid tag that
encodes the perturbation on to the response-capture bead; (h)
optionally converting the response of the cell to the perturbation
to a nucleic acid signal, wherein the response of the cell to the
perturbation is not a nucleic acid signal; and (i) sequencing the
nucleic acid tag attached to the response-capture bead, thereby
correlating the identity of the perturbation to the response of the
cell to the perturbation.
[0087] In still another aspect, the disclosure features a method
for perturbing a cell and capturing a response of the cell to the
perturbation by: (a) providing an array of picowells and a library
of functionalized perturbation beads, wherein the picowells are
capable of accommodating a single cell and a single functionalized
perturbation bead, wherein each functionalized perturbation bead
comprises a different plurality of substantially identical
releasable compounds and a plurality of nucleotide barcodes that
encodes the compounds, wherein the nucleotide barcodes are
functionalized barcodes capable of capturing cellular content of
the cell, wherein the cellular content of cell comprises cellular
response to the perturbations contained in the functionalized
perturbation beads; (b) capturing single cells into each picowell
of the picowell array; (c) capturing single functionalized
perturbation beads to the picowells containing single cells; (d)
releasing the compounds from the functionalized perturbation beads
and incubating the cells with the released compounds, wherein the
compounds between picowells have minimal diffusion; (e) lysing the
cells to release the cellular contents; (f) capturing one or more
components of the cellular content onto functionalized
oligonucleotides on the functionalized perturbation beads, wherein
the capturing comprises hybridization and enzymatic extension to
combine nucleotide barcodes with nucleic acid elements of the
cellular content, thereby forming a hybrid of the nucleotide
barcode and the nucleic acid element of the cellular content; and
(g) releasing the hybrid, collecting the hybrid from the library of
functionalized perturbation beads, and sequencing the hybrid,
thereby relating the perturbation to the cellular response to the
perturbation.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0088] FIG. 1. Concatenated-style bead. In concatenated-style bead,
the DNA barcode takes the form of all of the DNA barcode modules
connected to each other in a single chain, together with any other
nucleic acids that have functions, such as primer annealing sites,
as a spacer, or information on date of manufacture. The numbers on
this figure are not structure numbers. The numbers refer to the
sequence of "DNA barcode modules" in the DNA barcode.
[0089] FIG. 2. Orthogonal-style bead. In orthogonal-style bead, the
DNA barcode takes the form of all of the DNA barcode modules, where
the DNA barcode modules do not occur together in a single chain,
but instead occur separately linked to different positions on the
bead. The numbers on this figure are not structure numbers. The
numbers refer to the sequence of "DNA barcode modules" in the DNA
barcode.
[0090] FIGS. 3A-3I. Cleavable linkers, conditions for cleavage (UV
light or chemical), and cleavage products. Information from,
Yinliang Yang (2014) Design of Cleavable Linkers and Applications
in Chemical Proteomics. Technische Universitat Munchen Lehrstuhl
fur Chemie der Biopolymere. The alphabet letter to the left of each
linker is from this reference.
[0091] FIG. 4. Exemplary amino acid derivatives for the
compositions and methods of the present disclosure.
[0092] FIGS. 5A-5H. The photograph discloses increases in
degradation of a fusion protein, inside HeLa cells, with increasing
concentrations of added lenalidomide. Top: Expression of IKZF1/GFP
fusion protein. Bottom: Expression of mScarlett.RTM. control.
Lenalidomide was added at zero, 0.1, 1.0, or 10 micromolar.
[0093] FIGS. 6A-6H. The photograph discloses increases in
degradation of a fusion protein, inside HeLa cells, with increasing
concentrations of added lenalidomide. Top: Expression of IKZF3/GFP
fusion protein. Bottom: Expression of mScarlett control.
Lenalidomide was added at zero, 0.1, 1.0, or 10 micromolar.
[0094] FIG. 7. Methods and reagents for creating bead-bound DNA
barcode. The most accurate description of "DNA barcode" is the sum
of all of the information that is contained in the sum of all DNA
barcode modules. But for convenience, the term "DNA barcode" is
used herein to refer to the sum of all of the information of all of
the DNA barcode modules plus any additional nucleic acids that
provide information such as step number, or general type of
chemical monomers that make up the bead-bound compound, and plus
any additional nucleic acids that serve a function, such as linker,
sequencing primer binding site, hairpin with sequencing primer
binding site, or spacer. Where a DNA barcode is made, at least in
part, by way of click chemistry, the DNA barcode may include
residual chemical groups from the click chemistry reactions.
[0095] FIG. 8. Structure of Alexa Fluor.RTM. 488. A goal of this
figure is to identify the compound without having to resort to
using the trade name.
[0096] FIG. 9. Simplified diagram of bead-bound release-monitor.
The release-monitor provides the user with a measure of the
concentration of the soluble compound, following UV-induced release
of the compound from the bead. In a preferred embodiment, one type
of bead is dedicated to being a release-monitor, that is, this bead
does not also contain bead-bound compound and does not also contain
bead-bound DNA library. "PCL" is photocleavable linker.
[0097] FIG. 10. Detailed diagram of bead release-monitor.
[0098] FIG. 11. Chemical synthesis of bead release-monitor.
[0099] FIG. 12. Amine-functionalized bead with bifunctional linker,
where the linker includes a lysine residue.
[0100] FIG. 13. Steps of chemical synthesis of lenalidomide
modified with a first type of carboxyl group.
[0101] FIG. 14. Steps of chemical synthesis of lenalidomide
modified with a second type of carboxyl group.
[0102] FIG. 15. Steps of chemical synthesis of lenalidomide
modified with a third type of carboxyl group.
[0103] FIG. 16A, FIG. 16B, FIG. 16C. Lenalidomide analogues.
[0104] FIG. 17. Steps of chemical synthesis of a deoxycytidine
analogue suitable for click-chemistry synthesis of a DNA
barcode.
[0105] FIGS. 18A, 18B, and 18C. Caps for placing over the top of
picowells and for sealing the picowells. FIG. 18A shows active cap,
where compound is releasable by way of cleavable linker. FIG. 18B
shows another type of active cap, where a reagent such as an
antibody is bound. The bound reagent can be permanently linked, it
can be linked by a cleavable linker, or it can be bound by way of
hydrogen bonds and be releasable merely by exposure to the solution
in the picowell followed by diffusion away from the active cap and
into this solution. FIG. 18C shows a passive cap, which can be used
to absorb, adsorb, collect, or capture metabolites from the
solution in the picowell. The absorbed metabolites can subsequently
be analyzed.
[0106] FIGS. 19A, 19B, 19C, and 19D. FIG. 19A Picowell plate
without caps over the picowells. FIG. 19B. Picowell plate with a
cap over each picowell. FIG. 19C. Polyacrylamide solution being
poured over the picowell plate that has one cap securely fastened
over each picowell. The polyacrylamide then seeps into the porous
cap, solidifies, and forms a stable adhesion to each cap. FIG. 19D.
The solidified polyacrylamide "roof" is then peeled off from the
picowell plate, bringing with it each cap. The metabolites
transferred from the picowell solution and absorbed into each cap
can then be analyzed. Preferably, the solution that is poured over
the picowell plate and over the bead becomes a hydrogel, and
preferably the bead is made from a hydrogel.
[0107] In exclusionary embodiments, the present disclosure can
exclude a system, microtiter plate, microtiter plate with
microwells, nanowells, or picowells, and related methods, where at
least one well is capped, and where a liquid polymer solution is
poured over the plate and over the capped wells. Also, what can be
excluded is the above where the liquid polymer has polymerized to
form a solid polymer that adheres to each cap. Also, what can be
excluded is the method and resulting compositions, where the solid
polymer is torn away, removing with it the adhering caps.
[0108] FIG. 20. Map of circular plasmid used for integrating IKZF1
gene into genome of a cell. The plasmid is: IKZF1
mNEON-p2a-mScarlet-w3-2FB (9081 base pairs). IKZF1 encodes the
Ikarus protein.
[0109] FIG. 21. Map of circular plasmid used for integrating IKZF3
gene into genome of a cell. The plasmid is: IKZF3
mNeon-p2a-mScarlet-w3-2FB (9051 bp). IKZF3 encodes the Aiolos
protein.
[0110] FIG. 22. Chemical monomers (compounds 1-6) and their DNA
barcodes.
[0111] FIG. 23. Chemical monomers (compounds 7-10) and their DNA
barcodes.
[0112] FIG. 24. Chemical monomers (compounds 11-16) and their DNA
barcodes.
[0113] FIG. 25. Chemical monomers (compounds 17-21) and their DNA
barcodes.
[0114] FIG. 26. Chemical monomers (compounds 22-16) and their DNA
barcodes.
[0115] FIG. 27 Chemical monomers (compounds 27-30) and their DNA
barcodes.
[0116] FIG. 28. Sequencing a bead-bound DNA barcode. The figure
discloses intensity of fluorescent signal for each of five
consecutive bases, where the five consecutive bases are part of a
bead-bound DNA barcode.
[0117] FIG. 29. Stepped picowell.
[0118] FIGS. 30A-30F. Time course of release of the fluorophore
from the bead. This shows operation of the bead-bound release
monitor, acquisition of fluorescent data at t=0 seconds, t=1
seconds, t=11 seconds, and t=71 seconds.
[0119] FIGS. 31A-31B. Emission data resulting after catalytic
action of aspartyl protease on quencher-fluorophore substrate.
[0120] FIG. 32. Drawings of cross-section of picowells,
illustrating various steps.
[0121] FIGS. 33A-33F. Titration data showing how increase in UV
dose results in greater cleavage of fluorophore from the bead. In
layperon's terms, this shows how a more powerful swing of the axe
influences chopping the fluorophore from the bead (the power of the
UV does is measured in Joules per centimeter squared). The notation
"Exposure" refers only to a parameter when taking the photograph.
It is just exposure time, when taking the photograph (it does not
refer to exposure time of the light doing the cleaving, or to the
light doing the exciting).
[0122] FIG. 34. TAMRA concentration versus luminous flux. What is
shown is concentration of free TAMRA, following release after
exposure to UV light at 365 nm.
[0123] FIG. 35 provides a hand-drawings of the quencher-fluorophore
substrate, and of cleavage of this substrate by the enzyme, with
consequent inhibition of enzyme. Also shown is the molecular
structure of bead-bound pepstatin-A, and bead-bound Fmoc-valine
(negative control).
[0124] FIG. 36. Steps in preparing beads for use in eventual
capture of mRNA from lysed cells, with subsequent manufacture of
cDNA library. This figure also occurs in one of the Provisional
applications (Compositions and Method for Screening Compound
Libraries on Single Cells), from which priority of the present
application is claimed.
[0125] FIG. 37. Tagging cells with DNA barcode, where tagging is by
way of a lipid that embeds in the cell membrane. This figure also
occurs in one of the Provisional applications (Compositions and
Method for Screening Compound Libraries on Single Cells), from
which priority of the instant application is claimed.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0126] As used herein, including the appended claims, the singular
forms of words such as "a," "an," and "the" include their
corresponding plural references unless the context clearly dictates
otherwise. All references cited herein are incorporated by
reference to the same extent as if each individual patent, and
published patent application, as well as figures, drawings,
sequence listings, compact discs, and the like, was specifically
and individually indicated to be incorporated by reference.
ABBREVIATIONS
[0127] Table 1 provides abbreviations and non-limiting
definitions.
TABLE-US-00001 TABLE 1 Abbreviations and non-limiting definitions
ACN Acetonitrile AMPSO 3-[(1,1-dimethyl-2-hydroxyethyl)
amino]-2-hydroxypropanesulphonic acid. AMPSO is one of the "Good
buffers" ((1966). Hydrogen Ion Buffers for Biological Research.
Biochemistry. 5: 467-477). Aperture As used herein, the term
aperture is used herein to refer to a physical substance that
defines an opening and, more specifically, to the minimal amount of
physical substance that is capable of defining an opening. Without
implying any limitation, this minimal amount of physical substance
preferably takes the form of a ring-shaped section of a wall.
Without limitation, the aperture can be considered to be a
ring-shaped section of a wall, where the thickness of the section
is about 0.2 nm, about 0.5 nm, about 10 nm, about 20 nm, about 50
nm, about 100 nm, about 200 nm, about 500 nm, about 1 micrometer
(um), about 2 um, about 5 um, and so on, where this thickness
measurement is in the radial direction extending away from an axis,
and where the axis is defined by the opening. 1-AP 1-Azidopyrene
ATB Active tuberculosis Barcode The term "DNA barcode" can refer to
a polynucleotide that identifies a chemical compound in its
entirety while, in contrast, "DNA barcode module" can refer to only
one of the monomers that make up the chemical compound. A short
definition of a "DNA barcode module" is that it identifies a
chemical library monomer. However, a "DNA barcode module" can be
used to identify the history of making that particular monomer. A
longer definition of a "DNA barcode module" is as follows. Each of
the following chemical library monomers need to be identified by a
different "DNA barcode module." Even the first reaction and the
second reaction have the same reactants (A and B), a different DNA
barcode module is used, because the products are different (the
products being either C or D). Also, even though the first reaction
and the third reaction result in the same product (the product
being "C"), a different DNA barcode module is used, because the
reactants are different (the reactants being either A + B, or X +
Y). Reaction Condition A + B .fwdarw. C Reaction condition A, for
example, with methane solvent A + B .fwdarw. D Reaction condition
A, for example, with methylene chloride solvent X + Y .fwdarw. C
BiNAP takes the form of two naphthalene groups attached to each
other by way of a BiNAP carbon-carbon bond between the 1-carbon of
the first naphthalene and the 1-carbon of the second naphthalene.
Each naphthalene group also contains an attached PPh.sub.2 group,
where the PPh.sub.2 group is attached to the naphthalene's
2-carbon. PPh.sub.2 takes the form of a phosophate group, to which
is attached two phenyl groups. In BiNAP, the phosphate is situated
in between the naphthalene and the PPh.sub.2. BTPBB Bis-Tris
propane breaking buffer BTPLB Bis-Tris propane ligation buffer
BTPWB Bis-Tris propane wash buffer Cap A cap is an object that can
serve as a plug, a stopper, a seal, and the like, for placing in
stable contact with a microwell, nanowell, or picowell. The cap can
be spherical, ovoid, cubical, cubical with rounded edges,
pyramidal, pyramidal with rounded edges, and so on. Unless
specified otherwise, the stated shape is the shape prior to partial
insertion or prior to full insertion into the picowell. Preferably,
when in use the cap is partially inserted into the picowell to form
a seal. In some embodiments, the cap may be loosely set on top of
the picowell without any partial insertion. Compound The term
"compound" is used here, without implying any limitation, to refer
to a completed chemical that is synthesized by connecting a
plurality of chemical monomers to each other, by way of solid phase
synthesis on a bead. Generally, the term "compound" refers to the
completed chemical that is to be tested for activity by way of an
assay. The term "compound" is not intended to include any linkers
that mediate binding of the completed chemical to the bead, and is
not intended to include any protecting groups that are to be
cleaved off, though it is understood that a "compound" that has a
protecting group may have pharmaceutical activity. The term
"compound" is NOT used to refer to bead-bound chemicals where not
all of the chemical monomers have been connected. If the term
"compound" is used in some other context herein, the skilled
artisan will be able to determine if this description is relevant
or not. COMU 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)
dimethylamino-morpholino-carbenium hexafluorophosphate (CAS no.
1075198-30-9) Concatenated A DNA barcode that is "concatenated,"
takes the form where all of the DNA barcode nucleic acid modules
are part of the same polymer. When a bead contains a DNA barcode
taking the barcode concatenated form, all of the information from
all of the constituent DNA barcode modules are present on the
polymer that is attached to a single attachment site on the bead.
Concatenated DNA refers to "end-to-end ligation" or "end-to-end
joining" (Farzaneh (1988) Nucleic Acids Res. 16: 11319-11326; Boyer
(1999) Virology. 263: 307-312). In contrast, the word "catenated"
refers to two circles of DNA that are linked to each other as in a
chain (Baird (1999) Proc. Nat'l. Acad. Sci. 96: 13685-13690). CuAAC
Copper-catalyzed azide-alkyne cycloaddition CRB Click reaction
buffer DAF Diazofluorene DBCO Dibenzocyclooctyne DBU
1,8-diazabicyclo [5.4.0] undec-7-ene DCE 1,2-Dichloroethane DCM
Dichloromethane DESPS DNA encoded solid-phase synthesis DIC
Diisopropyl carbodiimide DIEA N,N'-diisopropylethylamine DMA
Dimethylacetamide DMAP 4-Dimethylaminopyridine DMF
Dimethylformamide DI Deionized DTT Dithiothreitol EDC
Ethyl-dimethylaminopropyl-carbodiimide ELISA Enzyme linked
immunosorbent assay FMOC 9-Fluorenylmethoxycarbonyl FMOC-PCL-
4-[4-[1-(9-Fluorenylmethyloxycarbonylamino)ethyl]-2-methyoxy-5-n-
itrophenoxy] OH butanoic acid (CAS No. 162827-98-7) Functional In
the context of a bead-bound DNA barcode, and in the context of
manufacturing a nucleic acid bead-bound DNA barcode, the term
"functional nucleic acid" refers to nucleic acids with an active
biochemical function (a function that takes advantage of hydrogen
bonds, of hydrophobic interactions, of hydrophilic interactions, of
interactions with enzymes, etc.). The function can be a spacer that
establishes a distance between a hydrophobic bead and a primer
binding site. The primer binding site preferably occurs in a
hydrophilic environment for supporting activity of DNA polymerase.
Also, the function can be a primer binding site, a hairpin bend, or
the annealing site for a "splint oligo." This is in contrast to
"informational nucleic acids," which store information (which
"encode") information on the identity of a corresponding chemical
monomer. HDNA Headpiece DNA HTS High throughput screening INA
5-Iodonaphthalene-1-azide LC Liquid chromatography LTB Latent
tuberculosis MDM2 Murine Double Minute 2 Mtt 4-Methyltrityl NCL
hits; NCL NCL refers to a mixture of sera from latent tuberculosis
patients (this accounts for the pool letter "L") and sera from
negative control, healthy human subjects (this accounts for the
letter "NC") NHS N-Hydroxysuccinimide. NHS chemistry can be used to
attach tetrazine to free amino groups of, for example, antibodies
(van Buggenum, Gerlach, Mulder (2016) Scientific Reports. 6: 22675.
Nucleic acid The term "nucleic acid" can refer to a single nucleic
acid molecule, or to modified nucleic acids, such as a nucleic acid
bearing a fluorescent tag. Also, the term "nucleic acid" can be
used to refer individual contiguous stretches of nucleotides within
a longer polynucleotide. Here, the term "nucleic acid" makes it
more convient to refer to these individual stretches within a
longer polynucleotide, for example, as when the polynucleotide
comprises a first nucleic acid that is a primer-binding site, a
second nucleic acid that is a DNA barcode module, and a third
nucleic acid that identifies the step number in a multi-step
pathway of synthesis. OP Oligo pair. Oligo pair can refer to a
reagent that takes the form of a slipped heteroduplex, for example,
an aqueous solution of a slipped heteroduplex. Orthogonal A DNA
barcode that is "orthogonal," takes a form where each of the DNA
barcode nucleic acid modules occupies a different attachment site
on the bead. When a bead contains a DNA barcode barcode taking the
orthogonal form, the acquisition of all of the information of a
compound's DNA barcode requires separately sequencing each of the
attached DNA barcode modules. In other words, with an "orthogonal"
nucleic acid barcode, each and every one of the DNA barcode modules
that makes up the DNA barcode is dispersed over different
attachment sites on the same bead. OSu (OSu is N-Hydroxysuccinimide
the same as NHS) OXYMA Ethyl 2-cyano-2-(hydroxyamino)acetate
Parallel The term "parallel" refers to the situation where chemical
monomers are covalently attached to a bead, one by one, to create a
bead-bound compound, and where nucleic acid barcode modules are
also covalently attached to the same bead, one by one, to create a
bead-bound nucleic acid barcode. The chemical reaction that
attaches each chemical monomer is not carried out at exactly the
same time as the reaction (chemical or enzymatic) that attaches
each nucleic acid barcode module. Instead, these two reactions are
staggered, so that the parallel synthesis involves first attaching
the chemical monomer, and then attaching the corresponding nucleic
acid barcode module. Alternately, the staggered reaction can
involve first attaching the nucleic acid and then attaching the
corresponding chemical monomer. What is corresponding in this
situation, is that each nucleic acid barcode module serves to
identify the chemical monomer that is attached in the same round of
parallel synthesis.
PCL Photocleavable linker PEG Polyethylene glycol PDMS
Polydimethylsiloxane qPCR Quantitative polymerase chain reaction
Picowell Without implying any limitation on the presend disclosure,
the term "picowell" can be used to refer to a well or cavity in a
plate that contains an array of picowells, for example, over 50,000
picowells, over 100,000 picowells, over 200,000 picowells, over
500, 000 picowells, and so on. Typically, the volume of a picowell
(not including the volume of any beads that might be in the
picowell), is about 0.2 picoliters (pL), about 0.5 pL, about 1.0
pL, about 2.0 pL, about 5.0 pL, about 10 pL, about 20 pL, about 30
pL, about 40 pL, about 50 pL, about 75 pL, about 100 pL, about 200
pL, about 300 pL, about 400 pL, about 500 pL, about 600 pL, about
700 pL, about 800 pL, about 1000 pL, about 10,000 pL, about 100,000
pL, about 1,000,000 pL, or in a volume range defined by any of the
above two values, for example, about 0.5 to 2.0 pL. The volumes for
any "nanowell" and "microwell" can be set as above (except with the
term "pico" replaced by nano or micro). Unless specified otherwise,
explicitly or by context, the present disclosure refers to
picowells (rather than to nanowells or microwells). RAM Rink Amide
RCA Rolling circle amplification RT Room temperature SPS Solid
phase synthesis Slipped Slipped heteroduplex structure takes the
form of a first strand of ssDNA and a second heteroduplex strand of
ssDNA, where a dozen nucleotides at the 5'-end of the first strand
of ssDNA structure are complementary to a dozen nucleotides at the
5'-end of the second strand of ssDNA, and where the first strand of
ssDNA is binds to the second strand of ssDNA by way of a dozen
complementary base pairings that involve the respective 5'-termini.
The number "dozen" is purely exemplary and is not limiting.
Alternatively, the slipped heteroduplex structure could be
maintained as a hybridized duplex, by way of complementary base
pairing at the 3'-end of the first strand of ssDNA and the 3'-end
of the second strand of ssDNA. The term "slipped heteroduplex
structure" can alternatively be called a "staggered heteroduplex
structure." The term "slipped" does not imply that the heteroduplex
is slippery (can shift position0 as might be the case with a duplex
formed when oligo[C] hybridizes to oligo[G], or when oligo[A]
hybridizes to oligo[T]. TB Tuberculosis TBE Tris borate EDTA TBAI
Tetrabutyl ammonium iodide. TBTA
Tris[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine TCEP
Tris(2-carboxyethyl)phosphine. Reducing agent that can cleave
disulfide bonds. TCO Trans-cyclooctene TEAA Triethylammonium
acetate TEV protease Tobacco Etch Virus protease TFA
Trifluoroacetic acid TID 3-(trifluoromethyl)-3-(m-iodophenyl)
diazirine TIPS Triisopropyl silane TM Temperature of melting TMP
2,4,6-Trimethylpyridine QSY7 Xanthylium,
9-[2-[[4-[[2,5-dioxo-1-pyrrolidinyl)oxy] carbonyl]-1-piperidinyl]
sulfonyl]phenyl]-3,6-bis(methylphenylamino)-, chloride (CAS No.
304014-12-8) TAMRA 5(6)Carboxytetramethyl rhodamine
[0128] Reagents, kits, enzymes, buffers, living cells,
instrumentation, and the like, can be acquired. See, for example,
Sigma-Aldrich, St. Louis, Mo.; Oakwood Chemical, Estill, S.C.;
Epicentre, Madison, Wis.; Invitrogen, Carlsbad, Calif.; ProMega,
Madison, Wis.; Life Technologies, Carlsbad, Calif.; ThermoFisher
Scientific, South San Francisco, Calif.; New England BioLabs,
Ipswich, Mass.; American Type Culture Collection (ATCC), Manassas,
Va.; Becton Dickinson, Franklin Lakes, N.J.; Illumina, San Diego,
Calif.; 10.times. Genomics, Pleasanton, Calif.
[0129] Barcoded gel beads, non-barcoded gel beads, and microfluidic
chips, are available from 1CellBio, Cambridge, Mass. Guidance and
instrumentation for flow cytometry is available (see, e.g.,
FACSCalibur.RTM., BD Biosciences, San Jose, Calif., BD FACSAria
II.RTM. User's Guide, part no. 643245, Rev.A, December 2007, 344
pages).
[0130] A composition that is "labeled" is detectable, either
directly or indirectly, by spectroscopic, photochemical,
fluorometric, biochemical, immunochemical, isotopic, or chemical
methods, as well as with methods involving plasmonic nanoparticles.
For example, useful labels include, .sup.32P, .sup.33P, .sup.35S,
.sup.14C, .sup.3H, .sup.125I stable isotopes, epitope tags,
fluorescent dyes, Raman tags, electron-dense reagents, substrates,
or enzymes, e.g., as used in enzyme-linked immunoassays, or
fluorettes (Rozinov and Nolan (1998) Chem. Biol. 5:713-728).
TABLE-US-00002 TABLE OF CONTENTS FOR DETAILED DESCRIPTION (I) Beads
(II) One bead one compound (OBOC) (III) Coupling nucleic acids to
beads (IV) DNA barcodes (V) Coupling chemical compounds to beads
(VI) Coupling chemical monomers to each other to make a compound
(VII) Split and pool synthesis and parallel synthesis (VIII)
Fabricating picowells (IX) Deposit beads into picowells (X)
Sequencing bead-bound nucleic acids in picowells (XI) Releasing
bead-bound compounds from the bead (XII Biochemical assays for
compounds (XIII) Cell-based assays for compounds (I) BEADS
[0131] The methods and compositions of the present disclosure use
beads, such as monosized TentaGel.RTM. M NH.sub.2 beads (10, 20,
30, etc., micrometers in diameter)-, standard TentaGel.RTM. amino
resins (90, 130, etc. micrometers in diameter), TentaGel
Macrobeads.RTM. (280-320 micrometers in diameter) (all of the above
from Rapp Polymere, 72072 Tubingen, Germany). These have a
polystyrene core derivatized with polyethylene glycol (Paulick et
al (2006) J. Comb. Chem. 8:417-426). TentaGel.RTM. resins are
grafted copolymers consisting of a low crosslinked polystyrene
matrix on which polyethylene glycol (PEG) is grafted. Thus, the
present disclosure provides beads or resins that are modified to
include one or both of a DNA barcode and a compound, where the
unmodified beads take the form of grafted copolymers consisting of
a low crosslinked polystyrene matrix on which polyethylene glycol
(PEG) is grafted.
[0132] TentaGel.RTM. is characterized as, "PEG chains of molecular
masses up to 20 kilo Dalton have been immobilized on functionalized
crosslinked polystyrenes. Graft copolymers with PEG chains of about
2000-3000 Dalton proved to be optimal in respect of kinetic rates,
mobility, swelling and resin capacity." (Rapp Polymere, Germany).
Thus, the present disclosure provides beads or resins that take the
form of graft copolymers with PEG chains of about 2000-3000
Daltons. Regarding swelling, Comellas et al provides guidance for
measuring the ability of a bead to swell, for example, when soaked
in DCM, DMF, methyl alcohol, water, or a buffer used in enzyme
assays (Comellas et al (2009) PLoS ONE. 4:e6222 (12 pages)). The
unit of swelling is milliliters per gram of bead.
[0133] In an alternate bead embodiment, the present disclosure uses
a resin with a PEG spacer is attached to the polystyrene backbone
via an alkyl linkage, and where the resin is microspherical and
monosized (TentaGel.RTM. M resin).
[0134] In yet an alternate bead embodiment, the present disclosure
uses a resin with a PEG spacer attached to the polystyrene backbone
via an alkyl linkage, where the resin type exists in two
bifunctional species: First, surface modified resins: the reactive
sites on the outer surface of the beads are protected orthogonally
to the reactive sites in the internal volume of the beads, and
second, hybrid resins: cleavable and noncleavable ligands are
present in this support--developed for sequential cleavage
(TentaGel.RTM. B resin).
[0135] Moreover, in another embodiment, the present disclosure uses
a resin where a PEG spacer is attached to the polystyrene backbone
via an alkyl linkage, and where the macrobead resin shows very
large particle diameters and high capacities (TentaGel.RTM. MB
resin). Also, the present disclosure uses a resin where the PEG
spacer is attached to the polystyrene backbone via a benzyl ether
linkage. This resin can be used for immunization procedures or for
synthesizing PEG modified derivatives (PEG Attached PEG-modified
compounds) (TentaGEl.RTM. PAP resin).
[0136] Moreover, the beads can be, HypoGel.RTM. 200 resins. These
resins are composites of oligoethylene glycol (MW 200) grafted onto
a low cross-linked polystyrene matrix (Fluka Chemie GmbH, CH-9471
Buchs, Switzerland).
[0137] In some embodiments amino functionalized polystyrene beads,
without PEG linkers, may be used, for instance, monosized
polystyrene M NH.sub.2 microbeads (5, 10, 20 etc., micrometers in
diameter, also from Rapp Polymere, 72072 Tubingen, Germany).
[0138] In some embodiments, compounds may be encapsulated within
pores or chambers or tunnels within the beads, without covalent
attachment to the beads. Compounds may be diffused into or forced
within such pores of the beads by various means. In some
embodiments the compounds may be loaded within the beads by
diffusion. In some embodiments, high temperature may be used to
swell the beads and load compounds within the beads. In some
embodiments, high pressure may be used to force compounds into the
beads. In some embodiments, solvents that swell the beads may be
used to load compound within the beads. In some embodiments, vacuum
or low pressure may be used to partition compounds into beads. In
some embodiments mild, or vigorous physical agitation may be used
to load compounds into beads.
[0139] In such embodiments where the compounds are loaded onto
beads without covalent attachment, compounds may be unloaded from
the bead by way of diffusion. In some embodiments, in a
non-limiting fashion, temperature, pressure, solvents, pH, salts,
buffer or detergent or combinations of such conditions may be used
to unload compounds out of such beads. In some embodiments the
physical integrity of the beads, for instance by uncrosslinking
polymerized beads, may be used to release compounds contained
within such beads.
[0140] In exclusionary embodiments, the present disclosure can
exclude any bead, and bead-compound complex, or any method, that
involves one of the above beads.
[0141] Beads of the present disclosure also include the following.
Merrifield resin (chloromethylpolystyrene); PAM resin
(4-hydroxymethylphenyl acetamido methyl polystyrene); MBHA resin
(4-methylbenzhydrylamine); Brominated Wang resin
(alpha-bromopriopiophenone); 4-Nitrobenzophenone oxime (Kaiser)
resin; Wang resin (4hydroxymethyl phenoxymethyl polystyrene; PHB
resin (p-hydroxybenzyl alcohol; HMPA resin (4-hydroxymethyl
phenoxyacetic acid); HMPB resin (4-hydroxymethyl-3-methoxy phenoxyl
butanoic acid); 2-Chlorotrityl resin; 4-Carboxytrityl resin; Rink
acid resin (4-[(2,4-dimethoxypehenyl) hydroxymethyl)
phenoxymethyl); Rink amide (RAM) resin "Knorr" resin
(4-((2,4-dimethylphenyl) (Fmox-amino)methyl) phenoxyalkyl); PAL
resin (5-[4-(Fmoc-amino) methyl-3,5-dimethoxyphenoxy]
valeramidomethyl polystyrene); Sieber amide resin
(9-Fmox-amino-xanthan-3-yl-oxymethyl); HMBA resin (hydroxymethyl
benzoic acid); 4-Sulfamoylbenzoyl resin "Kenner's safety catch"
resin (N-(4-sulfamoylbenzoyl) aminomethyhl-polystyrene); FMP-resin
(4-(4-formyl-3-methoxyphenoxy)-ethyl) (see, ChemFiles Resins for
Solid-Phase Peptide Synthesis Vol. 3 (32 pages) (Fluka Chemie GmbH,
CH-9471 Buchs, Switzerland).
[0142] Beads of the present disclosure further include the above
beads used as passive encapsulants of compounds (passively hold
compounds without covalent linkage to the compound), and further
comprising the following: unfunctionalized polystyrene beads;
silica beads; alumina beads; porous glass beads; polyacrylamide
beads; titanium oxide beads; alginate beads; ceramic beads; PMMA
(polymethylmethacrylate) beads; melamine beads; zeolite beds;
polylactide beads; deblock-copolymer micelles; dextran beads, and
others. Many of the beads listed in this paragraph may be purchased
from vendors such as Microspheres-Nanospheres, Cold Spring, N.Y.
10516, USA.
[0143] In addition to beads, vesicles or droplets may also be used
as vehicles for delivering compounds for some embodiments of the
present disclosure. Lipids, deblock-copolymers, tri-block
copolymers or other membrane forming materials may be used to form
an internal volume into which compounds may be loaded. Compounds
may be released from these encapsulated volumes by addition of
detergent, mechanical agitation, temperature, salt, pH or other
means. Water-in-oil droplet emulsions or oil-in-water droplet
emulsions are yet other means to passively encapsulate compounds
that may be delivered to assay volumes.
[0144] In all embodiments where passive encapsulation is used to
deliver compounds, DNA tags may also be loaded passively, or
alternatively, the DNA tags may be covalently attached to the
beads, vesicles or droplets.
[0145] In exclusionary embodiments, the present disclosure can
exclude any beads or resins that are made of any one the above
chemicals, or that are made of derivatives of one any one of the
above chemicals.
[0146] In embodiments, the beads can be spheroid and have a
diameter of about 0.1-1 micrometers, about 1-5 micrometers, about
1-10, about 5-10, about 5-20, about 5-30, about 10-20, about 10-30,
about 10-40, about 10-50, about 20-30, about 20-40, about 20-50,
about 20-60, about 50-100, about 50-200, about 50-300, about
50-400, about 100-200, about 100-400, about 100-600, about 100-800,
about 200-400, about 200-600, about 200-800 micrometers, and so
on.
[0147] Non-spheroid beads that are definable in terms of the above
values and ranges are also provided. For example, one of the axes,
or one of the primary dimensions (for example, a side) or one of
the secondary dimensions (for example, a diagonal) may comprise
values in the above ranges. In exclusionary embodiments, the
present disclosure can exclude any reagent, composition, system, or
method, that encompasses spheroid beads (or non-spheroid beads)
falling into one or more of the above values or ranges.
[0148] Chains of beads. In one embodiment, what is provided is a
plurality of bead dimers, where the bead-dimer takes the form of
two beads that are attached to each other, and where one bead
contains a plurality of attached nucleic acid barcodes (either
orthogonal nucleic acid modules, or concatenated nucleic acid
modules), and the other bead contains a plurality of attached
compounds, where all of the compounds are substantially related to
each other (or where all of the compounds are substantially
identical in chemical structure to each other). The bead dimer may
be synthesized by preparing the first bead that has the attached
compounds, separately preparing the second bead that has attached
nucleic acid barcodes, and then linking the two beads together. In
one aspect, the beads are attached to each other by a reversible
linker, and in another aspect, the beads are attached to each other
by a non-reversible linker.
[0149] Bead permeability. In embodiments, the present disclosure
provides beads with various ranges or degrees of permeability.
Permeability can be measured as the percentage of the volume of the
bead that is accessible by a solvent, where the unit of measurement
is percentage of the bead's surface that takes the form or pores,
or where the unit of measurement is percentage of the bead's
interior that takes the form of channels, networks, or chambers
that are in fluid communication with the surface (and exterior
medium) of the bead. The present disclosure can encompass porous
beads or, alternatively, can exclude porous beads.
[0150] U.S. Pat. No. 9,062,304 of Rothberg discloses a bead with an
exterior and with interior regions. What is shown is "internal
surfaces (pore surfaces)," and that "suitable pores will . . .
exclude larger molecules," and the option of "exploiting
differential functionalization of interior and exterior surfaces,"
and various pore diameters, and polymers such as poly(styrene
sulfonic acid) and polystyrene. FIG. 1 of Rothberg provides
pictures of surface of bead and pores of bead. U.S. Pat. No.
9,745,438 of Bedre provides transmission electron microscope image
of porous bead. U.S. Pat. No. 5,888,930 of Smith provides scanning
electron micrograph of cross-section of porous bead. What is shown
is sphereical bead with small pores on surface and large pores
inside, where bead is made from, e.g., polystyrene,
polyacrylonitrile, polycarbonate, cellulose, or polyurethane. U.S.
Pat. No. 5,047,437 of Cooke discloses sphereical
poly(acrylonitrile) copolymer pore morphology with skinless surface
(FIG. 1) and bead that has exterior skin on surface (FIG. 5). U.S.
Pat. No. 4,090,022 of Tsao discloses porous openings and internal
void spaces, of cellulose beads.
[0151] Each of the above-identified patents, including all of the
figures, is incorporated herein in its entirety, as though each was
individually incorporated by reference in its entirety.
[0152] Without implying any limitation, exterior surface of a bead
or microparticle can be determined by tightly wrapping the entire
bead or microparticle with an elastic film. The bead or
microparticle can be wrapped by way of a thought-experiment, or the
wrapped bead can be depicted by a drawing or photograph, or the
bead can be wrapped in reality. Without implying any limitation,
the exterior surface of the bead is that part of the bead that
physically contacts the wrapping.
[0153] For example, the present disclosure provides a bead with
pores accounting for at least 1%, at least 2%, at least 5%, at
least 10%, at least 15%, at least 20%, at least 30%, at least 40%,
of the surface area. Also, the present disclosure provides a bead
where the volume of the internal channels or networks accounts for
at least 1%, at least 2%, at least 5%, at least 10%, at least 15%,
at least 20%, at least 30%, at least 40%, at least 50%, at least
60%, at least 70%, at least 80%, of the total volume of the bead,
and where the internal channels or networks are in fluid
communication with the outside surface (and exterior medium) of the
bead.
[0154] Moreover, the present disclosure provides a bead with pores
accounting for less than 1%, less than 2%, less than 5%, less than
10%, less than 15%, less than 20%, less than 30%, less than 40%, of
the surface area. Also, the present disclosure provides a bead
where the volume of the internal channels or networks accounts for
less than 1%, less than 2%, less than 5%, less than 10%, less than
15%, less than 20%, less than 30%, less than 40%, less than 50%,
less than 60%, less than 70%, less than 80%, of the total volume of
the bead, and where the internal channels or networks are in fluid
communication with the outside surface (and exterior medium) of the
bead.
[0155] Iron-core beads. The present disclosure encompasses
iron-core beads or magnetic beads. These beads can be manipulated
with magnets to move them from one reaction vessel to another
reaction vessel, or from one container to another container.
Manipulations by robotics can be enhanced by using these beads.
Methods of manufacture and use of magnetic beads are available
(Szymonifka and Chapman (1994) Tetrahedron Letters. 36:1597-1600;
Liu, Qian, Xiao (2011) ACS Comb. Sci. 13:537-546; Alam, Maeda,
Sasaki (2000) Bioorg. Med. Chem. 8:465-473).
[0156] In exclusionary embodiments, the present disclosure can
exclude any bead, or any population of beads, where the bead or
population meets one of the above values or ranges.
[0157] Compound Loading into Beads
[0158] In many experiments it is advantageous to load
pre-synthesized compounds into beads, where the beads may be used
as vehicles for delivering the compounds to an assay. Many of the
standard techniques used for drug delivery to biological specimens
may be adapted to deliver compounds to assays (see, Wilczewska et
al (2012) Nanoparticles as drug delivery systems. Pharmacological
Reports. 64:1020-1037, Kohane DS (2007) Microparticles and
nanoparticles for drug delivery. Biotechnol. Bioeng. 96: 203-209,
Singh et al (2010) Microencapsulation: A promising technique for
controlled drug delivery. Res Pharm Sci. 5: 65-77).
[0159] In such embodiments where pre-synthesized compounds are
loaded into beads, the compounds may be held in traditional 96, 385
or 1536 well microtiter plates. To these plates, beads may be
added, into which the compounds get loaded by diffusion or by other
active loading methods. In preferred embodiments, the beads chosen
for impregnation have pore sizes or percolation geometries that
prevent immediate emptying of the compounds when removed from the
mother solution. The diffusion out of the beads may be enhanced by
heat, pressure, additives or other stimulants, if needed. In some
embodiments, the compound-laden beads may be capped in a manner
that prevents leakage of the internal contents until triggered by
an external impulse. One method for capping the exteriors of porous
beads involves adding lipids or amphiphilic molecules to the
bead-compound solution, such that the cavities exposed to the
surface of the beads get sealed by a bilayer formed by the
amphiphilic molecules. In some embodiments preformed vesicles may
be mixed with the drug laden beads, such that upon agitating, the
vesicles rupture and the membranes reform over the surface of the
drug-laden beads, thereby sealing them. Methods to perform such
bead sealing are described (see, Tanuj Sapra et al (2012) Nature
Scientific Reports volume 2, Article No.: 848). Further
experimental protocols to seal silica beads are available in the
report release by Sandia Laboratories by Ryan Davis et al,
Nanoporous Microbead Supported Bilayers: Stability, Physical
Characterization, and Incorporation of Functional Transmembrane
Proteins, SAND2007-1560, and the method bSUM, described by Hui
Zheng et al (bSUM: A bead-supported unilamellar membrane system
facilitating unidirectional insertion of membrane proteins into
giant vesicles) in J. Gen. Physiol. (2016) 147: 77-93.
[0160] In some embodiments utilizing pre-synthesized compounds,
beads are generated from the compounds by addition of appropriate
reagents, for instance by adding lipids or di-block copolymers
followed by agitation, whereby vesicles are formed containing the
compounds in their interior or within the bilayer membrane. In some
embodiments, the compounds may be pushed through a microfluidic T
junction to create aqueous phase droplets in an oil phase, where
the compounds are contained within the aqueous phase or at the
interface between the aqueous phase and the oil phase. In some
embodiments, the droplets formed may further be polymerized,
creating hydrogels, that are more rugged and stable to handling
than unpolymerized aqueous phase droplets. Droplet-based
encapsulation and assays are disclosed by, Oliver et al (2013)
Droplet Based Microfluidics, SLAS Discovery Volume: 19 issue: 4,
page(s): 483-496. Sol-gel encapsulation process may also be
employed to encapsulate compounds within beads. Formation of
sol-gel beads is described in, Sol-gel Encapsulation of
Biomolecules and Cells for Medicinal Applications, Xiaolin Wang et
al (2015) Current Topics in Medicinal Chemistry. 15: 223.
[0161] One Bead One Compound (OBOC)
[0162] Methods used to manufacture combinatorial libraries involve
three steps, (1) Preparing the library; (2) Screening the compounds
in the library, and (3) Determining the structure of the compounds,
for example, of all of the compounds or only of the compounds that
provided an interesting result with screening (see, Lam et al
(1997) The One-Bead-One-Compound Combinatorial Library Method.
Chem. Rev. 97:411-448). An advantage of synthesizing compounds by
way of a bead-bound synthesis, is that the compound can be made
rapidly by the "split-and-pool" method.
[0163] OBOC combined with an encoding strategy. Another feature of
OBOC is that each bead can include, not only a compound but also an
encoding strategy. Where bead-bound nucleic acids are used for
encoding a compound that is bound to the same bead, the term
"encoding" does NOT refer to the genetic code. Instead, the term
"encoding" means that the user possesses a legend, key, or code,
that correlates each of many thousands of short nucleic acid
sequences with a single bead-bound compound.
[0164] A dramatic variation of using a bead that bears bead-bound
compounds and bead-bound nucleic acids, where the nucleic acids
encode the associated compounds, is as follows. The dramatic
variation is to manufacture a library of conjugates, where each
member of the library takes the form of a conjugate of a small
molecule plus a DNA moiety, where the DNA moiety encodes the small
molecule). This conjugate is soluble and is not bead-bound. After
screening with a cell or with a purified protein, the conjugate
remains bound to the cell or purified protein, thereby enabling
isolation of the conjugate and eventual identifying the compound by
sequencing the conjugated nucleic acid (see, Satz et al (2015)
Bioconjugate Chemistry. 26:1623-1632).
[0165] Here, as in most of this patent document, the term "encode"
does not refer to the genetic code, but instead it refers to the
fact that the researcher uses a specific nucleic acid sequence to
indicate a specific, known structure of a compound that is attached
to it.
[0166] As an alternative to using an encoding strategy, such as the
use of a DNA barcode, a bead that screens positive (thereby
indicating a compound that screens positive) can be subjected to
Edman degradation or to mass spectrometry to identify the
bead-bound compound (see, Shih et al (2017) Mol. Cancer Ther.
16:1212-1223). If the bead-bound compounds are peptides, then MALDI
mass spectrometry can be used for direct determination of the
sequence of a positively-screening peptide compound. Direct
sequencing is possible, because simultaneous cleavage and
ionization occur under laser irradiation (Song, Lam (2003) J. Am.
Chem. Soc. 125:6180-6188).
[0167] One fine point in performing split-and-pool synthesis of a
combinatorial library, is that the compound can be manufactured so
that all of the compounds share a common motif. This strategy has
been described as the, "generation of a library of motifs rather
than a library of compounds" (see, Sepetov et al (1995) Proc. Natl.
Acad. Sci. 92:5426-5430; Lam et al, supra, at 418).
[0168] To provide a typical example of a large bead, the bead can
be 0.1 mm in diameter and it can hold about 10.sup.13 copies of the
same compound (Lam et al, supra). Following preparation of a
library of bead-bound compounds, each bead can be used in
individual assays, where the assays measure biochemical activity
or, alternatively, a binding activity. Assays can be "on-bead"
assays or, alternatively, the compound can be severed from the bead
and used in solution-phase assays (Lam et al, supra).
[0169] Parameters of any type of bead include its tendency to swell
in a given assay medium, whether the bead's polymer is hydrophobic
or hydrophilic, the identity of the attachments sites on the bead
for attaching each compound, the issue of whether a spacer such as
polyethylene glycol (PEG) is used to provide some separation of
each compound from the bead's surface, and the internal volume of
the bead.
[0170] Regarding the need to attach compounds to the bead, but at a
distance far away from the bead's hydrophobic surface, Lam et al,
supra, discloses that polyoxyethylene-grafted styrene
(TentaGel.RTM.) has the advantage that the functionalizable group
is at the end of a polyoxyethylene chain, and thus far away from
the hydrophobic polystyrene. Beads that possess a water-soluble
linker include TentaGel and polydimethylacrylamide bead
(PepSyn.RTM. gel, Cambridge Research Biochemicals, Northwitch,
UK).
[0171] The parameter of internal volume can provide an advantage,
where there is a need to prevent interactions between the
bead-bound DNA barcode and the target of the bead-bound compound.
To exploit this advantage, the bead can be manufactured so that the
DNA barcode is situated in the inside of the bead while, in
contrast, the compound that is being screened is attached to the
bead's surface (Lam et al, supra, at pages 438-439). This advantage
of internal volume may be irrelevant, where the bead-bound compound
is attached by a cleavable linker, and where assays of the compound
are conducted only on compounds that are cleaved and released.
[0172] Appell et al, provide a non-limiting example of
spit-and-pool method for synthesizing a chemical library followed
by screening to detect active compounds (Appell et al (1996) J.
Biomolecular Screening. 1:27-31). Library beads are placed, one
into each well, in an array of wells on a first microwell plate,
nanowell plate, or picowell plate. Beads are exposed to light, in
order to cleave about 50% of the bead-bound compounds, releasing
them into solution in the well. Released compound is then
transferred to a second microwell plate, and subjected to assays
for detecting wells that contain active compounds, thereby
identifying which beads in the first plate contain bead-bound
compounds that are active. Then, "[o]nce an active [compound] is
identified from a single bead, the bead is recovered and decoded,
thus yielding the synthetic history and . . . structure of the
active compound" (Appell et al, supra).
[0173] For cell-based screening assays that screen for bead-bound
compounds, Shih et al provide a novel type of bead (Shih et al
(2017) Mol. Cancer Ther. 16:1212-1223). This novel type of bead
contains a bead-bound compound that is a member of a library of
"synthetic death ligands against ovarian cancer." The bead is also
decorated with biotin, where two more chemicals are added that
create a sandwich, and where the sandwich maintains adhesion of the
cell to the bead. The sandwich includes a streptavidin plus
biotin-LXY30 complex. This sandwich connects the bead to LXY30's
receptor, which happens to be a well-known protein on the cell
surface, namely, an integrin. The method of Shih et al, supra,
resulted in the discovery of a new molecule ("LLS2") that can kill
cancer cells. The above method uses bead-bound compounds, where the
compounds bind to cells (even though the compound is still
bead-bound). Cho et al created a similar one-bead-one-compound
library, where the compound being screened was sufficient to bind
to cells (without any need for the above-described sandwich) (Cho
et al (2013) ACS Combinatorial Science. 15:393-400). The goal of
the Cho et al, report was to discover RGD-containing peptides that
bind to integrin that is expressed by cancer cells. The
above-disclosed reagents and methods are useful for the present
disclosure.
[0174] Coupling Nucleic Acids to Beads (Orthogonal Style;
Concatenated Style)
[0175] One way to get oriented to the topic of concatenated
barcodes and orthogonal barcodes, is to note advantages that one
has over the other. An advantage of orthogonal barcoding over
concatenated barcoding, is as follows. With attachment of each
monomer of a growing chemical compound, what is attached in
parallel is a DNA barcode module. With concatenated barcoding, if
attachment of any given module is imperfect (meaning, that not all
of the attachments sites was successfully coupled with a needed
module), then the sequence of the completed barcode will not be
correct. The statement "not be correct" means that imperfect
coupling means that chunks may be missing from wad was assumed to
be the completed, correct DNA barcode. Here, the completed barcode
sequence will contain a mistake, due to failure of attachment of
all of the modules. In contrast, with orthogonal barcoding each
individual module gets covalently bound to its own unique
attachement site on the bead. And where once a module gets attached
to a given site on the bead, no further modules will be connected
to the module that is already attached.
[0176] The present disclosure provides reagents and methods for
reducing damage to bead-bound DNA barcodes, and for reducing damage
to to partially synthesized bead-bound DNA barcodes. Each DNA
barcode module, prior to attaching to a growing bead-bound DNA
barcode, can take the form of double stranded DNA (dsDNA), where
this dsDNA is treated with a DNA cross-linker such as mitomycin-C.
After completion of the synthesis of the DNA barcode in its dsDNA
form, this dsDNA is converted to ssDNA. Conversion of dsDNA to
ssDNA can be effected where one of the DNA strands has a uracil (U)
residue, and where cleavage of the DNA at the position of the
uracil residue is catalyzed by uracil-N-glycosidase (see, FIG. 5 of
Ser. No. 62/562,905, filed Sep. 25, 2017. Ser. No. 62/562,905 is
incorporated herein by reference in its entirety). The above refers
to damage that is inflicted on the growing DNA barcode by reagents
used to make the bead-bound chemical compound.
[0177] Another method for reducing damage to bead-bound DNA
barcodes, and for reducing damage to partially synthesized DNA
barcodes, is by synthesizing the DNA barcode in a double stranded
DNA form, where each of the DNA barcode modules that are being
attached to each other takes the form of dsDNA, and where each of
the two strands is stabilized by way of a DNA headpiece. For
eventual sequencing of the completed DNA barcode, one of the
strands is cleaved off from the DNA headpiece and removed. The
above refers to damage that is inflicted on the growing DNA barcode
by reagents used to make the bead-bound chemical compound (where
this chemical compound is a member of the chemical library).
[0178] Yet another method for reducing damage to bead-bound DNA
barcodes, is to synthesize the DNA barcode in a way that
self-assembles to form a hairpin, and where this DNA barcode
self-assembles to that the first prong of the hairpin anneals to
the second prong of the hairpin.
[0179] Where the DNA barcode being synthesized takes the form of
double stranded DNA (dsDNA), solvents such as DCM, DMF, and DMA can
denature the DNA barcode. The above methods and reagents can
prevent denaturation.
[0180] As stated above, the term "DNA barcode" can refer to a
polynucleotide that identifies a chemical compound in its entirety
while, in contrast, "DNA barcode module" can refer to only one of
the monomers that make up the chemical compound.
[0181] Another method for reducing damage to bead-bound DNA
barcodes, and for reducing damage to partially synthesized DNA
barcodes, is to use double stranded DNA (dsDNA) and to seal the
ends of this dsDNA by way of 7-aza-dATP and dGTP.
[0182] In alternate embodiments, the method can use an intermediate
between "concatenated DNA barcoding" and "orthogonal DNA
barcoding," where this intermediate involves blocks of DNA
barcodes, that is, where each block contains two DNA modules, or
contains three DNA modules, or contains four DNA modules, or
contains five DNA modules, and the like (but does not contain all
of the DNA modules that identify the full-length compound).
[0183] FIG. 1 discloses an exemplary and non-limiting diagram of
the CONCATENATED structured bead. The bead contains a plurality of
DNA barcodes (each made of DNA barcode modules) and a plurality of
compounds (each made of chemical library monomers). For ease in
speaking, the term "DNA barcode" may be used to refer to the
polymer that includes all of the nucleic acids that are a "DNA
barcode module," as well as all of the nucleic acids that provide
some function. The function can be an annealing site for a
sequencing primer, or the function can be used to identify a step
in chemical synthesis of the bead-bound compound. FIG. 1 also shows
bead-bound compounds, where each compound is made of several
chemical library members, and where each chemical library member is
represented by a square, circle, or triangle. FIG. 1 shows that
each DNA barcode module is numbered, consecutively, from 1 to 8,
where these numbers correspond to the respective eight shapes
(squares, circles, triangles). For clarity, nucleic acids that
serve a function (and do not represent or "encode" any particular
chemical unit) are not shown in the figure.
[0184] FIG. 2 discloses an exemplary and non-limiting embodiment of
the ORTHOGONAL structured bead. The bead contains a plurality of
DNA barcodes (each made of DNA barcode modules), but each DNA
barcode module is attached to a separate linking site on the bead.
The entire DNA barcode consists of eight DNA barcode modules, which
in the figure are numbered 1-8. When the information from a
particular DNA barcode is read, and then used to identify the
chemical compound that is bound to the same bead, one must perform
DNA sequencing on each of the separately attached DNA barcode
modules. In FIG. 2, the bead also contains a plurality of attached
chemical compounds, each with eight units, as shown by the eight
shapes (circles, squares, triangles).
[0185] In FIG. 2, for clarity, functional nucleic acids that are
attached to each DNA barcode module is not shown. Of course, each
of the DNA barcode modules needs to have a nucleic acid that
identifies the position of the chemical library monomer in the
completed, full-length compound. For the example shown in FIG. 2,
the position needs to be first, second, third, fourth, fifth,
sixth, seventh, or eighth.
[0186] In one embodiment, the chemical monomer is first attached
and then, after that, the corresponding DNA barcode module is
attached. In an alternative embodiment, the DNA barcode module is
first attached, and then the corresponding chemical monomer is
attached. Also, a procedure of organic synthesis can be followed
that sometimes uses the "one embodiment" and sometimes uses the
"alternative embodiment." In yet another alternative embodiment,
the present method provides block-wise addition of a block of
several chemical monomers which is attached to the bead, in
parallel with attachment of a block of several DNA barcode
modules.
[0187] In exclusionary embodiments, what can be excluded is
reagents, compositions, and methods that used block-wise addition
of chemical monomers, of DNA barcode modules, or of both chemical
monomers and DNA barcode modules, to a bead.
[0188] This concerns nucleic acids that may be present in the
bead-bound polynucleotide, including nucleic acids that "encode" or
serve to identify monomers of a bead-bound compound. In
exclusionary embodiments, the present disclosure can exclude a
nucleic acid that encodes a "step-specific DNA sequencing primer
site." In this situation, for each chemical monomer that is present
in a compound, there is a corresponding DNA barcode module, where
each DNA barcode module is flanked by at least one corresponding
primer-binding site, that is, "a step-specific DNA sequencing
primer site." Also, what can be excluded is a nucleic acid that
encodes or designates a particular step in the chemical synthesis
of a compound, such as step 1, step 2, step 3, or step 4.
[0189] Moreover, the present disclosure can include a nucleic acid
that functions as a spacer. For example, as spacer can create a
distance, along a polynucleotide chain, between a first site that
is a sequencing primer annealing site and a second site that
identifies a chemical monomer. Also, the present disclosure can use
a nucleic acid that reiterates or confirms the information provided
by another nucleic acid. Also, the present disclosure can use a
nucleic acid that encodes a PCR primer binding site. A PCR primer
binding site can be distinguished from a sequencing primer, because
a polynucleotide with a PCR primer binding site has two PCR primer
binding sites, and because both of these sites are designed to have
the same melting point (melting point when the PCR primer is
annealed to PCR primer binding site).
[0190] In exclusionary embodiments, the present disclosure can
exclude a nucleic acid that functions as a spacer, or solely as a
spacer. Also, the present disclosure can exclude a nucleic acid
that reiterates or confirms the info provided by another nucleic
acid. Moreover, the present disclosure can exclude a nucleic acid
that serves as a PCR primer binding site, and can exclude a nucleic
acid that serves as a binding site for a primer that is not a PCR
primer.
[0191] Additionally, the present disclosure can exclude a nucleic
acid that identifies the date that a chemical library was made, or
that identifies a step in chemical synthesis of a particular
compound, or that serves as a primer annealing sequence.
[0192] Dedication of sequencing primers to a particular DNA barcode
module. The present disclosure provides a DNA barcode that contains
DNA barcode modules and one or more sequencing primer annealing
sites. Each DNA barcode module may have its own, dedicated,
sequencing primer binding site. Alternatively, one particular
sequencing primer binding site may be used for sequencing two,
three, four, five, 6, 7, 8, 9, 10, or more consecutive DNA barcode
modules, as may exist on the bead-bound DNA barcode.
[0193] The following describes the situation where each DNA barcode
module has its own dedicated sequencing primer binding site. The
present disclosure provides a bead-bound concatenated barcode
comprising a primer binding site capable of binding a DNA
sequencing primer, wherein said primer binding site is capable of
directing sequencing of one or more of the 1.sup.st DNA barcode
module, the 2.sup.nd DNA barcode module, the 3.sup.rd DNA barcode
module, the 4.sup.th DNA barcode module, the 5.sup.th DNA barcode
module, and the 6.sup.th DNA barcode module, and wherein the primer
binding site is situated 3-prime to the 1.sup.st DNA barcode module
with no other DNA barcode module in between the 1.sup.st DNA
barcode module and the primer binding site, 3-prime to the 2.sup.nd
DNA barcode module with no other DNA barcode module in between,
3-prime to the 3.sup.rd DNA barcode module with no other DNA
barcode module in between, 3-prime to the 4.sup.th DNA barcode
module with no other DNA barcode module in between, 3-prime to the
5.sup.th DNA barcode module with no other DNA barcode module in
between, or 3-prime to the 6.sup.th DNA barcode module with no
other DNA barcode module in between.
[0194] Encoding sequences and sequences complementary to encoding
sequences. The present disclosure can encompass any one, any
combination of, or all of the encoding sequences disclosed above,
or elsewhere, in this document. In exclusionary embodiments, what
can be excluded are any one, any combination of, or all of the
encoding sequences disclosed above, or elsewhere, in this document.
What can also be included or can be excluded are double stranded
nucleic acids that encode any one, any combination of, or all of
the encoding sequences described above, or elsewhere, in this
document.
[0195] Orthogonal-Style DNA Barcode (Each DNA Barcode Module
Attached to Separate Location on Bead)
[0196] Synthesis of orthogonal-style bead. With orthogonal
synthesis, each DNA module gets covalently attached to a separate
site on the bead, and where the result is that the entire DNA
barcode is contributed by a plurality of DNA modules. Where the DNA
barcode has the orthogonal structure, none of the DNA barcode
modules are attached to each other--instead each and every one of
the DNA barcode molecules has its own bead-attachment site that is
dedicated to that particular DNA barcode module.
[0197] Nucleic acid identifying the synthesis step number for each
DNA barcode module. In embodiments, the orthogonal DNA barcode
includes a short nucleic acid that identifies the first step of
compound synthesis. For this embodiment, with the parallel
attachment of the first chemical monomer and the first DNA barcode
module, the first DNA barcode module actually takes the form of
this complex of two nucleic acids: [SHORT NUCLEIC ACID THAT MEANS
"STEP ONE"] connected to [FIRST DNA BARCODE MODULE]. All of the
nucleotides of this complex are in-frame with each other and can be
read in a sequencing assay, but the first short nucleic acid may
optionally be attached to the first DNA barcode module by way of a
spacer nucleic acid.
[0198] The following continues the above description of the
orthogonal DNA barcode. The orthogonal DNA barcode includes a short
nucleic acid that identifies the second step of compound synthesis.
For this embodiment, with the parallel attachment of the second
chemical monomer and the second DNA barcode module, the second DNA
barcode module actually takes the form of this complex of two
nucleic acids: [SHORT NUCLEIC ACID THAT MEANS "STEP TWO"] connected
to [SECOND DNA BARCODE MODULE]. All of the nucleotides of this
complex are in-frame with each other and can be read in a
sequencing assay, but the second short nucleic acid may optionally
be attached to the second DNA barcode module by way of a spacer
nucleic acid.
[0199] The above-described method is repeated for the third,
fourth, fifth, sixth, seventh, eighth, ninth, tenth, and up to the
last of the DNA barcode modules and up to the last of the chemical
monomers, for any given bead. The above-method can be followed when
using split-and-pool synthesis, for creating DNA barcodes and
chemical compounds that are bead-bound.
[0200] The orthogonal structure provides the following advantage
over the concatenated structure. With concatenated synthesis (all
DNA barcode modules attached to each other in one, continuous
polymer) it is the case that failure to achieve synthesis any of
the intermediates coupling steps can ruin the meaning of the
concatenated DNA barcode that is eventually completed. In contrast,
with orthogonal synthesis (each and every one of the DNA barcode
modules attached to a dedicated site on the bead), failure to
attach any of the DNA barcode modules will only result in an empty
attachment site on the bead, and will not ruin the meaning of any
of the other attached DNA barcode modules. In a preferred
embodiment, each attached DNA barcode module includes an attached,
second nucleic acid, where this second nucleic acid identifies the
step (the step during the parallel synthesis of DNA barcode and
chemical compound).
[0201] For orthogonal synthesis, it is acceptable for all of the
attachment sites on the bead to be used up (sites for attaching the
growing chemical library member). However, for orthogonal
synthesis, the chemical reaction needs to be designed so that the
entire population of attachment sites on the bead is only partly
used up, with attachment of the first of many DNA barcode modules.
The following provides optional limits for using up sites during
chemical synthesis of an orthogonal barcode. For the non-modified
bead, the total number of sites available for attaching a DNA
barcode module is 100%.
[0202] Extent of using up attachment sites on a given bead, with
synthesis of an orthogonal-configured bead (regarding the 1.sup.st
DNA barcode). The following concerns attaching the first DNA
barcode module. In embodiments, with attachment of the first DNA
barcode module, about 5%, about 10%, about 20%, about 30%, about
40%, or about 50% of the DNA barcode attachments sites on the bead
are used up. In other embodiments, less than about 2%, less than
about 5%, less than about 10%, less than about 20%, less than about
30%, less than about 40%, or less than about 50% of the DNA barcode
attachments sites on the bead are used up. In still other
embodiments, with attachment of the first DNA barcode module,
between 2-4%, between 2-6%, between 2-8%, between 2-10%, between
2-12%, between 2-14%, between 2-16%, between 2-18%, between 2-20%,
between 10-20%, between 10-25%, between 10-30%, between 10-35%,
between 10-40%, of the DNA barcode attachment sites are used
up.
[0203] Regarding limits, with attaching the last of the DNA barcode
modules that make up a particular DNA barcode, less than 20% of the
sites are used up, less than 30%, less than 40%, less than 50%,
less than 60%, less than 70%, less than 80%, less than 90%, less
than 95%, or less than 98% of the sites are used up.
[0204] Exclusionary embodiments can exclude beads or methods that
match any of the above values or ranges. Also, exclusionary
embodiments can exclude beads or methods that fail to match any of
the above values or ranges.
[0205] The following concerns polymers that comprises one or more
nucleic acids, each being a DNA barcode, as well as polymers that
comprise two or more nucleic acids, where some of the nucleic acids
have a biochemical function such as serving as a primer-annealing
site or as a spacer, and where other nucleic acids have an
informational function and are DNA barcodes. In exclusionary
embodiments, the present disclosure can exclude a DNA barcode that
includes a DNA crosslinking agent such as psoralen. Also, what can
be excluded is a DNA barcode with a primer binding region with a
higher melting temperature (or a lower melting temperature) than a
DNA barcode module. This temperature can be merely "higher" or
"lower" or it can be at least 2 degrees C. higher, at least 4
degrees C. higher, at least 6 degrees C. higher, at least 8 degrees
higher, or at least 2 degrees C. lower, at least 4 degrees C.
lower, at least 6 degrees C. lower, at least 8 degrees lower.
[0206] Also what can be excluded is a method for making a DNA
barcode that uses DNA ligase. Also, what can be excluded is a DNA
barcode and methods for making, that comprise a hairpin (ssDNA bent
in a loop, so that one portion of the ssDNA hybridizes to another
portion of the same ssDNA). Additionally, what can be excluded is a
composition with a nucleic acid hairpin, where the nucleic acid
hairpin is covalently closed, for example, with a chemical linker.
Moreover, what can be excluded is a DNA barcode that is covalently
linked, either directly to a "headpiece," or indirectly to
"headpiece" (indirectly by way of covalent binding to one or more
chemicals that reside in between DNA barcode and the
headpiece).
[0207] In other exclusionary embodiments, what can be excluded is a
bead-bound DNA barcode, where the completed DNA barcode does not
comprise any double stranded DNA (dsDNA), but only comprises single
stranded DNA (ssDNA).
[0208] Extent of using up attachment sites on a given bead, with
synthesis of an orthogonal-configured bead (regarding the 2.sup.nd
DNA barcode). The following concerns attaching the second DNA
barcode module. In embodiments, with attachment of the second DNA
barcode module (for the creation of the orthogonal configured
bead), about 5%, about 10%, about 20%, about 30%, about 40%, or
about 50% of the remaining free DNA barcode attachments sites on
the bead are used up. In other embodiments, less than about 5%,
less than about 10%, less than about 20%, less than about 30%, less
than about 40%, or less than about 50% of the remaining free DNA
barcode attachments sites on the bead are used up. In still other
embodiments, with attachment of the first DNA barcode module,
between 2-4%, between 2-6%, between 2-8%, between 2-10%, between
2-12%, between 2-14%, between 2-16%, between 2-18%, between 2-20%,
between 10-20%, between 10-25%, between 10-30%, between 10-35%,
between 10-40%, of the remaining free DNA barcode attachment sites
are used up.
[0209] Exclusionary embodiments can exclude beads or methods that
match any of the above values or ranges. Also, exclusionary
embodiments can exclude beads or methods that fail to match any of
the above values or ranges.
[0210] The above embodiments, as well as the above exclusionary
embodiments, can also be applied to a method with attaching a third
DNA module barcode, or with attaching a fourth DNA module barcode,
or with attaching a fifth DNA barcode module, and so on.
[0211] Concatenated-Style DNA Barcode (all DNA Barcode Modules
Reside in One Chain or Polymer, where the Entire Chain or Polymer
is Attached to One Location on the Bead).
[0212] Synthesis of bead-bound concatenated-style DNA barcode. The
present disclosure provides a bead-bound concatenated-style DNA
barcode, where the bead contains a plurality of concatenated-style
DNA barcodes, and where most or nearly all of the plurality of
concatenated-style DNA barcodes have essentially the same
structure. The concatenated-style DNA barcode can contain one or
more DNA barcode modules, where the ordering of these DNA barcode
modules (from the bead-attachment terminus to the distal terminus)
along the entire DNA barcode, takes the same order as the time that
the bead-bound concatenated-style DNA barcode is synthesized. Also,
the ordering of these DNA barcode modules along the entire DNA
barcode, takes the same order as the time that a corresponding
chemical library monomer is coupled to the growing bead-bound
compound.
[0213] The concatenated-style DNA barcode can comprise, in this
order, a linker that is used to couple the entire
concatenated-style DNA barcode to the bead. Also, it can comprise,
in this order, a 1.sup.st DNA barcode module, a 1.sup.st annealing
site, a 2.sup.nd DNA barcode module, a 2.sup.nd annealing site, a
3.sup.rd DNA barcode module, and a 3.sup.rd annealing site.
[0214] One ordering of sequencing primer hybridizing site in a
bead-bound DNA barcode. In sequencing primer hybridizing site
embodiments, the concatenated-style DNA barcode can comprise, in
this order, a linker, a 1.sup.st DNA barcode module, a 1.sup.st
annealing site, a 1.sup.st sequencing primer binding site, a
2.sup.nd DNA barcode module, a 2.sup.nd annealing site, a 2.sup.nd
sequencing primer binding site, a 3.sup.rd DNA barcode module, a
3.sup.rd annealing site, and a 3.sup.rd sequencing primer binding
site, and so on.
[0215] Another ordering of the sequencing primer hybridizing site,
as it occurs in a bead-bound DNA barcode. In another sequencing
primer hybridizing site embodiment, the concatenated-style DNA
barcode can comprise, in this order, a linker, a 1.sup.st DNA
barcode module, a 1.sup.st sequencing primer binding site, 1.sup.st
annealing site, a 2.sup.nd DNA barcode module, a 2.sup.nd
sequencing primer binding site, a 2.sup.nd annealing site, a
3.sup.rd DNA barcode module, a 3rd sequencing primer binding site,
and 3.sup.rd annealing site, and so on.
[0216] The term "annealing site." The term "annealing site" is used
to refer to an annealing site that is part of a splint
oligonucleotide (splint oligo) and also to refer to the
corresponding bead-bound annealing site that resides on a growing
bead-bound DNA barcode. The skilled artisan understands that the
"annealing site" on the splint oligo does not possess the same DNA
sequence as the corresponding "annealing site" on the growing
bead-bound DNA barcode. In other words, the skilled artisan
understands that one sequence is complementary to the other
sequence. Therefore, it is of no consequence that, for the
descriptions herein, both annealing sites have the same name. In
other words, it is of no consequence that the 2.sup.nd annealing
site on a splint oligo is disclosed as one that hybridizes to the
2.sup.nd annealing site on growing bead-bound DNA barcode.
[0217] Synthesis in blocks. In an alternative embodiment, the
growing compound and the growing sequence of DNA barcode modules
can be synthesized in blocks. For example, a block consisting of
2-chemical library units can be attached to a bead in parallel with
attaching a block consisting of corresponding 2-DNA barcode
modules. Similarly, a block consisting of 3-chemical library units,
can be attached to a bead in parallel with attaching a block
consisting of a corresponding 3-DNA barcodes. Block synthesis
involving blocks of four, blocks of five, blocks of six, blocks of
seven, blocks of eight, blocks of nine, blocks of ten, and so on,
are also provided. Each of these block transfer embodiments can
also be excluded by the present disclosure. The blockwise transfer
of DNA barcode monomers can be done orthogonally, with unique
attachment points for receiving each of successive blocks of DNA
barcode momers. Alternatively, blockwise transfer of DNA barcode
monomers can be done to produce a concatemer structure (all DNA
barcode modules occurring as only one continuous, linear
polymer).
[0218] Also, during split-and-pool synthesis in parallel of the
bead-bound DNA barcode and the bead-bound compound, synthesis of
can occur in blocks. The block can take the form of two or more
chemical library monomers, and the block can take the form of two
or more DNA barcode modules.
[0219] Location of split-and-pool synthesis. Split-and-pool
synthesis can be used for the parallel synthesis of bead-bound
compounds and bead-bound concatenated DNA barcode. Also,
split-and-pool synthesis can be used for the parallel synthesis of
bead-bound compounds and bead-bound orthogonal DNA barcode. The
concatenated DNA barcode can be made by way of the "splint oligo"
method. Alternatively, concatenated DNA barcode can be made by way
of click chemistry. Also, a combination of the "splint oligo"
method and click chemistry can be used. Split-and-pool synthesis
can occur in a 96 well plate, where each well has a floor made of a
0.25 micrometer filter. Under normal gravity conditions, aqueous
solutions do not flow through this filter. However, suction can be
applied to remove any aqueous solutions from all of the 96 wells,
for example, where there is a need to replace a first aqueous
solution with a second aqueous solution. This suction method is
used when the bead is exposed to a first set of reagents, or when
the first set of reagents needs to be rinsed out, or when the first
set of reagents needs to be replaced by a second set of reagents. A
manifold is used to hold the 96 well plate (Resprep VM-96 manifold)
and a pump can be used to draw fluid out the bottom of every filter
(BUCHI Vac V-500 pump). The 96 well plate with the filter bottom
was, AcroPrep Advance 96 well, 350 uL, 0.45 um, REF 8048 (Pall
Corp., Multi-Well Plates, Ann Arbor, Mich.).
[0220] Distance from primer annealing site to a DNA barcode module.
For the purpose of sequencing a bead-bound DNA barcode, that is,
for the goal of sequencing all of the DNA barcode modules that form
the DNA barcode, a polynucleotide comprising a first nucleic acid
that is an annealing site for a sequencing primer, and a second
nucleic acid that is a DNA barcode module, the first nucleic acid
can be immediately upstream of the second nucleic acid.
Alternatively, the first nucleic acid can be upstream of the second
nucleic acid, where the first and second nucleic acids are
separated from each other by one, two, three, four, five, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, or more nucleotides, or by about one,
about two, about three, about about four, about five, about 6,
about 7, about 8, about 9, about 10, about 11, about 12, about 13,
about 14, or about 15 nucleotides. The separation can be with
nucleic acids that merely serve as a spacer or, alternatively, the
separation can be with a third nucleic acid that encodes
information, such as step number in a multi-step pathway of organic
synthesis, or the nume of a class of chemical compounds, or a
disease that might be treatable by the bead-bound compound, or the
date, or a lot number, and so on.
[0221] Synthesis of Bead-Bound Concatenated DNA Barcode Using Click
Chemistry
[0222] Click chemistry can be used for the step-by-step synthesis
of a DNA barcode. Here, what can be coupled is a first DNA barcode
module directly to a bead, or a first DNA barcode module to a
bead-bound linker.
[0223] Also, what can be coupled is a polynucleotide taking the
form of a first nucleic acid that is a 1.sup.st DNA barcode module
attached to a second nucleic acid that is a 1.sup.st sequencing
primer binding site. This sequencing primer binding site allows the
operator to determine the sequence of the 1.sup.st DNA barcode
module.
[0224] To provide another example, what can be coupled is a
2.sup.nd DNA barcode module directly to a bead-bound 1.sup.st DNA
barcode module. Alternatively, what can be coupled is a
polynucleotide taking the form of a first nucleic acid that is a
2.sup.nd DNA barcode module attached to a second nucleic acid that
is a 2.sup.nd sequencing primer binding site. This sequencing
primer binding site allows the operator to determine the sequence
of the 2.sup.nd DNA barcode module. If there is read-through to the
1.sup.st DNA barcode module, then what can be determined is the
sequence of both of these DNA barcode modules.
[0225] To provide yet another example, what can be coupled is a
polynucleotide comprising a first nucleic acid that is a 1.sup.st
DNA barcode module, and a second nucleic acid that identifies the
step in a multi-step parallel synthesis of the DNA barcode and of
the compound. Also, or alternatively, the second nucleic acid can
identify the general class of compounds that are being made by the
split-and-pool synthesis. Also, or alternatively, the second
nucleic acid can identify a disease that is to be treated by the
compounds to be screened. Also, the second nucleic acid can
identify the date, or the name of the chemist, and so on.
[0226] A preferred method for synthesizing the DNA barcode is shown
below, where the same cycle of reactions is used with progressive
attachment of each DNA barcode module.
[0227] Step 1. Provide a bead with an attached TCO group. In actual
practice, the bead will have hundreds or thousands of identically
attached TCO groups, where each TCO group is attached to a
different site on the bead. Also, in actual practice, a large
number of beads will be simultaneously modified by click chemistry,
with employment of the split-and-pool method.
[0228] Step 2. Add [tetrazine]-[first DNA barcode module]-[azide]
to the bead, and allow the TCO group condense with the tetrazine
group. The result is the following construct:
BEAD-TCO-tetrazine-first DNA barcode module-azide. In actual
practice, this construct does not include any TCO or tetrazine, but
instead has the condensation product that is created when TCO
condenses with tetrazine.
[0229] Step 3. Optional wash.
[0230] Step 4. Add DBCO-TCO in order to cap the azide and to create
a TCO terminus The result is the following structure:
[0231] BEAD-TCO-tetrazine-first DNA barcode
module-azide-DBCO-TCO
[0232] Step 5. Optional wash.
[0233] Step 6. Add the following reagent, which attaches the second
DNA barcode module. Attachment is to the distal terminus of the
growing DNA barcode. The reagent is:
[0234] [tetrazine]-[second DNA barcode module]-[azide] to the bead,
and allow the TCO group condense with the tetrazine group. The
result is the following construct:
[0235] BEAD-TCO-tetrazine-first DNA barcode
module-azide-DBCO-TCO-[tetrazine]-[second DNA barcode
module]-[azide]
[0236] The above scheme includes a cycle of steps for the stepwise
addition of more and more DNA barcode modules, where these
additions are in parallel with additions of more and more chemical
monomers. As stated elsewhere, this "parallel" synthesis can
involve attaching a chemical monomer followed by attaching a DNA
barcode module that identifies that monomer or, alternatively,
attaching a DNA barcode module followed by attaching a chemical
monomer that is identified by that particular chemical monomer.
[0237] Compounds for Click-Chemistry Synthesis of DNA Barcode
[0238] FIG. 17 discloses the chemical synthesis of a compound
suitable for connecting a deoxycytidine reside (dC) during the
synthesis of a DNA barcode module and, ultimately, the entire DNA
barcode. The starting material is N4-acetyl-2'-deoxy-5'-O-DMT
cytidine. The abbreviation "DMT" stands for 4,4-dimethoxytrityl.
The final product of this multi-step pathway of organic synthesis
bears a cytosine moiety, a triphosphate group, and a propargyl
group that is attached to the 3'-position of the ribose group. The
propargyl group is used for click chemistry, where it condenses
with an azide group to produce a covalent bond. After condensing,
the result is that a residual chemical (never naturally present in
nucleic acids), occurs as a "scar" from the click chemistry that
had been performed. What is available is DNA polymerases that can
be used for sequencing-by-synthesis of DNA barcodes made by click
chemistry, and where the DNA polymerases can move across the scars,
and where the scars do not cause sequencing errors. TBAI is
tetrabutyl ammonium iodide.
[0239] Synthesis of Concatenated Configuration DNA Barcode
[0240] In the following description, DNA barcode modules are
assembled in a row in order to create the DNA barcode. However, in
the in-text diagrams that are shown below, the term "DNA barcode"
is used instead of "DNA barcode module," in order to make the
in-text diagrams fit on the page. FIG. 7 illustrates the same steps
as shown here, but with additional details, such as diagrams of
beads. A reiterated sequence of reactions can be used for adding
each additional DNA barcode module.
[0241] Option of creating a DNA barcode that includes a terminal
nucleic acid that encodes DNA hairpin. This concerns a DNA barcode
that includes, at the 3-prime end, a nucleic acid that possesses an
annealing site for a sequencing primer, a bend taking the form of
about four bases that are not base-paired, and a sequencing primer
that is capable of bending around and forming base pairs with the
sequencing primer annealing site. To repeat, the sequencing primer
anneals to the sequencing primer annealing site, where the actual
sequencing reaction begins at the 3'-teminus of the annealed
sequencing primer.
[0242] When it is time to perform a final step in synthesizing a
DNA barcode, and when the final DNA barcode module is to be coupled
to the growing bead-bound DNA barcode, the "splint oligo" can
include a sequence that encompasses a DNA hairpin (the DNA hairpin
including, in this order, an annealing site for the sequencing
primer, several nucleotides that do not base pair with each other
or with any nearby sequences of bases, and a sequencing primer).
After annealing the "splint oligo," then DNA polymerase and dNTPs
are added, where polymerization occurs at the 3'-end of the growing
DNA barcode, where what gets polymerized using the splint oligo as
a template is, in order: (1) Annealing site for sequencing primer;
(2) Bend in the hairpin taking the form of four or five
deoxyribonucleotides that do not base pair with teach other; and
(3) Sequencing primer.
[0243] Reversible terminator group at the 3'-end of the hairpin
sequencing primer. The present disclosure provides reagents,
compositions, and methods, for attaching a pre-formed complex of a
nucleotide/reversible terminator group, to the 3'-terminus of the
annealed sequencing primer. Reversible terminator group is an
optional component of the hairpin sequencing primer, where it is to
be part of a bead-bound DNA barcode.
[0244] STEP 1. At the start, we have a bead situated in a picowell,
where the bead bears a coupled polynucleotide, and where the 5'-end
of the polynucleotide is coupled to the bead, optionally, with a
linker. FIG. 7 shows that the bead-bound polynucleotide comprises a
1.sup.st DNA barcode and a 1.sup.st annealing site. The linker can
be made of a nucleic acid, or it can be made of some other
chemically. Preferrably, the linker is hydrophobic, and preferably
the linker separates the bead-bound DNA barcode from the
hydrophobic polystyrene bead, for example, a TentaGel.RTM.
bead.
[0245] For convenience in writing, a 1.sup.st annealing site that
is part of a bead-bound DNA barcode and a 1.sup.st annealing site
that is part of a soluble "splint oligo" are both called "1.sup.st
annealing site," even though they do not have the same sequence of
bases (instead, the sequence of bases are complementary to each
other, where the result is tha the splint oligo can hybridize to
the 1.sup.st annealing site on the bead-bound growing DNA barcode,
thereby serving as a template for DNA polymerase to extend the
bead-bound DNA barcode by copying what is on the splint oligo.
[0246] Also, for convenience in writing, a 2.sup.nd annealing site
that is part of a bead-bound DNA barcode and a 2.sup.nd annealing
site that is part of a soluble "splint oligo" are both called,
"2.sup.nd annealing site," even though they do not have the same
sequence (but instead have complementary bases).
[0247] The bead-bound growing DNA barcode, from the 5'-end to the
3'-end, may contain the nucleic acids in the following order:
[0248] Bead-/1.sup.st DNA Barcode/1.sup.st Annealing Site/
[0249] Alternately, the bead-bound growing DNA barcode, from the
5'-end to the 3'-end, may include a nucleic acid that encodes the
step number, where the bead-bound growing DNA barcode has nucleic
acids in the following order:
[0250] Bead-/1.sup.st DNA Barcode/Nucleic Acid Encoding Step
Number/1.sup.st Annealing Site/
[0251] Alternatively, the bead-bound growing DNA barcode can
include a nucleic acid that is a functional nucleic acid (a
sequencing primer annealing site), as shown below:
[0252] Bead-/1.sup.st DNA Barcode/Sequencing Primer Annealing
Site/1.sup.st Annealing Site/
[0253] What is not shown in these in-text diagrams is an optional
linker that mediates coupling of the DNA barcode to the bead. The
linker can take the form of a nucleic acid, or it can be made of
some other organic chemical.
[0254] STEP 2. Add a soluble splint oligonucleotide (splint oligo),
where this splint oligo comprises a 1.sup.st annealing site and a
2.sup.nd DNA barcode module, and a 2.sup.nd annealing site.
[0255] FIG. 7 also illustrates the step where the hybridized splint
oligo is used as a template, where DNA polymerase catalyzes the
attachment to the bead-bound growing DNA barcode of the 2nd DNA
barcode module and the 2' annealing site. FIG. 7 shows the
enzymatic product where DNA polymerase catalyzes uses the splint
oligo as a template, resulting in the bead-bound DNA barcode
growing by a bit longer (growing by covalent attachment of the
2.sup.nd DNA barcode and the 2.sup.nd annealing site. What is shown
immediately below in the text, is the complex of the splint oligo
that is hybridized to the bead-bound growing DNA barcode:
[0256] Bead-/1.sup.st DNA Barcode/1.sup.st Annealing Site/
[0257] . . . 1.sup.st Annealing Site/2.sup.nd DNA Barcode/2.sup.nd
Annealing Site
[0258] To reiterate some information shown in FIG. 7, what is shown
immediately below is the splint oligo:
[0259] "1.sup.st annealing site/2.sup.nd DNA Barcode/2.sup.nd
Annealing Site"
[0260] STEP 3. DNA polymerase and dNTPs are added to extend the
bead-bound DNA barcode. Shown below is the bead-bound growing DNA
barcode, with the splint oligo still hybridized to it, and where
the bead-bound growing barcode is longer than before, because what
is now attached to it is a nucleic acid that is the "2.sup.nd DNA
barcode module" and a nucleic acid that is the "2.sup.nd annealing
site." FIG. 7 also illustrates this step. The splint oligo is shown
underneath the bead-bound growing barcode:
[0261] Bead-/1.sup.st DNA Barcode/1.sup.st Annealing Site/2.sup.nd
DNA Barcode/2.sup.nd Annealing Site
[0262] . . . 1.sup.st Annealing Site/2.sup.nd DNA Barcode/2.sup.nd
Annealing Site
[0263] Step 4. Wash away the splint oligo. The splint oligo can be
encouraged to dissociate from the bead-bound growing barcode by
heating, that is, by heating the entire picowell plate, for
example, to about 60 degrees C., about 65 degrees C., about 70
degrees C., about 75 degrees C., about 80 degrees C., for about ten
minutes or, alternatively, by adding dilute NaOH to the picowell
array, and then neutralizing.
[0264] Step 5. Add a second splint oligo which, after hybridizing
to the bead-bound growing splint oligo, can be used as a template
for mediating DNA polymerase-catalyzed attachment of a 3rd DNA
barcode and a 3rd annealing site. This second splint oligo, which
is a soluble reagent, is shown below (but it is not shown in FIG.
7):
[0265] 2.sup.nd Annealing Site/3.sup.rd DNA Barcode/3.sup.rd
Annealing Site/
[0266] Step 6. Allow this oligonucleotide to anneal to the
corresponding bead-bound "2.sup.nd annealing site," and allow DNA
polymerase to extend the bead-bound oligonucleotide, so that it
contains a complement to the: "3.sup.rd DNA barcode/3.sup.rd
annealing site/
[0267] Step 7. Wash away the second splint oligo.
[0268] Step 4. Add the following splint oligo (this particular
addition is not shown in FIG. 7).
[0269] 3rd Annealing Site/4th DNA Barcode/4th Annealing Site/
[0270] This soluble oligonucleotide has a nucleic acid that can
anneal to the "3.sup.rd annealing site" of the bead-bound
oligonucleotide. Once annealed, DNA polymerase with four dNTPs are
employed and used for extending the bead-bound oligonucleotide to
encode yet another DNA barcode module (the 4.sup.th DNA barcode).
The above cycle of steps is repeated, during the entire
split-and-pool procedure that creates, in parallel, the library of
chemical compounds and the associated DNA barcodes, where each DNA
barcode is associated with a given compound (where each DNA barcode
informs us of the history of chemical synthesis of the associated
compound). The above cycle of steps is stopped, when the chemical
synthesis of the library of compounds has been completed. With the
completed bead-bound, DNA barcoded chemical library in hand, the
beads can then be dispensed into microwells of a microwell
array.
[0271] The DNA barcode for each bead also constitutes a DNA barcode
that associated with each microwell. The DNA barcode allows
identification of the bead-bound compound. The sequencing method of
the present disclosure occurs inside the microwell while the bead
is still inside the microwell. In exclusionary embodiments, the
present disclosure can exclude any sequencing method and can
exclude exclude any reagents used for sequencing, where sequencing
is not performed on a DNA tempate that is bead-bound, or where
sequencing is not performed on a bead-bound DNA template that is
siutated inside a microwell.
[0272] Annealing sites for sequencing primer. In one embodiment,
each DNA barcode module in a completed DNA barcode is operably
linked and in frame with its own sequencing primer annealing site,
thus providing the operator with the ability to conduct separate
sequencing procedures on each DNA barcode module (in this
embodiment, it is preferred that each DNA barcode module is also
operably linked with its own nucleic acid that identifies (encodes)
the step in synthesis of the entire DNA barcode.
[0273] In another embodiment, each DNA barcode has only one
sequencing primer annealing site, where this can be situated at or
near the 3'-terminus of the bead-bound DNA barcode, and where the
sequencing primer itself can be soluble, added to the picowell, and
then hybridized to the sequencing primer annealing site.
Alternatively, where the sequencing primer is to be part of a DNA
hairpin, this DNA hairpin is added by way of a "splint oligo" at
the final step in creating the bead-bound DNA barcode. FIG. 7 does
not show any annealing sites for any sequencing primers.
[0274] Nucleic Acids Coupled to Beads by Way of the 3'-Terminus of
the Nucleic Acid
[0275] While various embodiments disclosed in this invention
pertain to coupling DNA to a bead by way of the DNA's 5'-end, in
other embodiments, DNA such as a DNA barcode or a DNA tag, can be
coupled to the bead by way of their 3'-end. The 3'-hydroxyl group
of DNA might be reactive under certain chemical synthesis
conditions (e.g. Mitsunobu transformations), rendering the 3'-end
damaged and unable to participate in extension, ligation or other
steps. Thus DNA tags may be attached to beads via their 3'-ends to
prevent unwanted chemical reactions and to prevent damage to the
DNA barcodes.
[0276] Exclusionary embodiments regarding bead-bound DNA barcodes
of the present disclosure. What can be excluded is any bead,
microparticle, microsphere, resin, or polymeric composition of
matter, wherein the concatenated DNA barcode is linked to the bead
by way of a photocleavable linker or by way of a cleavable
linker.
[0277] What can be excluded is any bead, microparticle,
microsphere, resin, or polymeric composition of matter, that does
not include both of the following: (1) Concatenated DNA barcode
that is coupled to a first position on the bead, (2) A compound
that is coupled to a second position on the bead, and wherein the
first position is not the same as the second position. In a
preferred embodiment, this "compound" is made of a plurality of
chemical library monomers.
[0278] What can be excluded is any bead, microparticle,
microsphere, resin, or polymeric composition of matter, that does
not have an exterior surface (or exterior surfaces) and also an
interior surface (or interior surfaces, or interior regions), and
where the bead does not comprise at least 10,000 substantially
identical concatenated DNA barcodes that are coupled to the bead,
and wherein at least 90% of the at least 10,000 substantially
identical concatenated DNA barcodes are coupled to the exterior
surface. In other words, what can be excluded is any bead where at
least 90% of the coupled concatenated DNA barcodes are not coupled
to the exterior surface.
[0279] What can be excluded is any bead, microparticle,
microsphere, resin, or polymeric composition of matter, that is
made substantially of polyacrylamide or that contains any
polyacrylamide.
[0280] What can be excluded is any bead, microparticle,
microsphere, hydrogel, resin, or polymeric composition of matter,
that contains a promoter, such as a T7 promoter, or that contains a
polyA region, or that contains a promoter and also a polyA
region.
[0281] Method with only one cycle of annealing/polymerization, to
produce a bead-bound DNA barcode with two DNA barcode modules. The
present disclosure encompasses systems, reagents, and methods,
where the bead-bound DNA barcode includes only one
annealing/polymerization step. This embodiment is represented by
the following diagrams, where the first diagram shows annealing of
the splint oligo, and the second diagram shows filling-in using DNA
polymerase. The end-result is a bead-bound DNA barcode that
contains two DNA barcode modules. In this particular procedure, the
bead-bound starting material can optionally include linker (but
preferably not any cleavable linker), optionally a nucleic acid
that encodes information other than identifying a chemical
compound, and optionally a functional nucleic acid, such as a
sequencing primer or a DNA hairpin. The two diagrams are shown in
the text (see, immediately below):
[0282] Bead-/1.sup.st DNA Barcode/1.sup.st Annealing Site/
[0283] . . . 1.sup.st Annealing Site/2.sup.nd DNA Barcode/2.sup.nd
Annealing Site
[0284] Bead-/1.sup.st DNA Barcode/1.sup.st Annealing Site/2.sup.nd
DNA Barcode/2.sup.nd Annealing Site
[0285] . . . 1.sup.st Annealing Site/2.sup.nd DNA Barcode/2.sup.nd
Annealing Site
[0286] Method with two cycles of annealing/polymerization, to
produce a bead-bound DNA barcode that has three DNA barcode
modules. The present disclosure encompasses bead-bound
compositions, systems, and methods, where two different split
oligos are used (first splint oligo; second splint oligo). In this
situation, the first splint oligo comprises the structure: 1.sup.st
annealing site/2.sup.nd DNA barcode/2.sup.nd annealing site, and
where the second splint oligo comprises the structure: 2.sup.nd
annealing site/3.sup.rd DNA barcode/3.sup.rd annealing site.
[0287] Method with three cycles of annealing/polymerization, to
produce a bead-bound DNA barcode that has four DNA barcode modules.
The present disclosure encompasses bead-bound compositions,
systems, and methods, where three different split oligos are used
(first splint oligo; second splint oligo, third splint oligo). In
this situation, the first splint oligo comprises the structure:
1.sup.st annealing site/2.sup.nd DNA barcode/2.sup.nd annealing
site, and where the second splint oligo comprises the structure:
2.sup.nd annealing site/3.sup.rd DNA barcode/3.sup.rd annealing
site, and where the third splint oligo comprises the structure:
3.sup.rd annealing site/4.sup.th DNA barcode/4.sup.th annealing
site.
[0288] Method with four cycles of annealing/polymerization, to
produce a bead-bound DNA barcode that has five DNA barcode modules.
The present disclosure encompasses bead-bound compositions,
systems, and methods, where four different split oligos are used
(first splint oligo; second splint oligo, third splint oligo,
fourth splint oligo). In this situation, the first splint oligo
comprises the structure: 1.sup.st annealing site/2.sup.nd DNA
barcode/2.sup.nd annealing site, and where the second splint oligo
comprises the structure: 2.sup.nd annealing site/3.sup.rd DNA
barcode/3.sup.rd annealing site, and where the third splint oligo
comprises the structure: 3.sup.rd annealing site/4.sup.th DNA
barcode/4.sup.th annealing site, and where the fourth splint oligo
comprises the structure: 4.sup.th annealing site/5.sup.th DNA
barcode/5.sup.th annealing site,
[0289] Embodiments with a plurality of steps of
annealing/polymerization, to produce a bead-bound DNA barcode that
has a plurality of DNA barcode modules. The present disclosure
encompasses bead-bound compositions, systems, and methods, relating
to concatenated barcodes, that uses only one splint oligo (making a
2-module DNA barcode), that uses only two splint oligos (making a
3-module DNA barcode), that uses only three splint oligos (making a
4-module DNA barcode), that uses only four splint oligos (making a
5-module DNA barcode), that uses only five splint oligos (making a
6-module DNA barcode), that uses only six splint oligos (making a
7-module DNA barcode), and so on.
[0290] What is encompassed is bead-bound compositions, systems, and
methods, that uses at least one splint oligo, at least two splint
oligos, at least three splint oligos, at least four splint oligos,
at least five splint oligos, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 11, at least 12, at last 13, at
least 14, at least 20 splint oligos, or less than 20, less than 15,
less than 10, less than 8, less than 6, less than 4, less than 3,
less than 2 splint oligos. These numbers refer to the splint oligo
itself, as well as to the number of the step of adding the splint
oligo, and also to the numbering of the DNA module that is added to
the growing bead-bound DNA barcode.
[0291] Reducing Damage to DNA Barcodes
[0292] Reducing damage by using orthogonal DNA barcodes (instead of
concatenated DNA barcodes). One way to get oriented to the topic of
concatenated DNA barcodes and orthogonal DNA barcodes, is to note
advantages that one has over the other. An advantage of orthogonal
barcoding over concatenated barcoding, is as follows. With
attachment of each monomer of a growing chemical compound, what is
attached in parallel are chemical library monomers to create a
chemical library, and DNA barcode modules to create a completed and
full-length DNA barcode.
[0293] With concatenated barcoding, if attachment of any given
module is imperfect (meaning, that not all of the attachments sites
were successfully coupled with a needed module), then the sequence
of the completed barcode will not be correct. The statement "not be
correct" means that imperfect coupling resulted in chunks that were
missing, where the user had assumed that the completed product was
a completed and correct DNA barcode. Here, the completed DNA
barcode sequence will contain a mistake, due to failure of
attachment of all of the DNA modules. In contrast, with orthogonal
barcoding each individual DNA module gets covalently bound to its
own unique attachment site on the bead. And where once a DNA module
gets attached to a given site on the bead, no further DNA modules
need to get coupled to the DNA modules that are already coupled to
the bead.
[0294] Reducing damage by using cross-linkers. The present
disclosure provides reagents and methods for reducing damage to
bead-bound DNA barcodes, and for reducing damage to to partially
synthesized bead-bound DNA barcodes. Each DNA barcode module, prior
to attaching to a growing bead-bound DNA barcode, can take the form
of double stranded DNA (dsDNA), where this dsDNA is treated with a
DNA cross-linker such as mitomycin-C. After completion of the
synthesis of the DNA barcode in its dsDNA form, this dsDNA is
converted to ssDNA. Conversion of dsDNA to ssDNA can be effected
where one of the DNA strands has a uracil (U) residue, and where
cleavage of the DNA at the position of the uracil residue is
catalyzed by uracil-N-glycosidase (see, FIG. 5 of Ser. No.
62/562,905, filed Sep. 25, 2017. Ser. No. 62/562,905 is
incorporated herein by reference in its entirety). The above refers
to damage that is inflicted on the growing DNA barcode by reagents
used to make the bead-bound chemical compound.
[0295] Reducing damage by using double stranded DNA (dsDNA) for
making DNA barcodes. Another method for reducing damage to
bead-bound DNA barcodes, and for reducing damage to partially
synthesized DNA barcodes, is by synthesizing the DNA barcode in a
double stranded DNA form, where each of the DNA barcode modules
that are being attached to each other takes the form of dsDNA, and
where each of the two strands is stabilized by way of a DNA
headpiece. For eventual sequencing of the completed DNA barcode,
one of the strands is cleaved off from the DNA headpiece and
removed. The above refers to damage that is inflicted on the
growing DNA barcode by reagents used to make the bead-bound
chemical compound (where this chemical compound is a member of the
chemical library).
[0296] Reducing damage by including a hairpin. Yet another method
for reducing damage to bead-bound DNA barcodes, is to synthesize
the DNA barcode in a way that self-assembles to form a hairpin, and
where this DNA barcode self-assembles to that the first prong of
the hairpin anneals to the second prong of the hairpin.
[0297] Where the DNA barcode being synthesized takes the form of
double stranded DNA (dsDNA), solvents such as DCM, DMF, and DMA can
denature the DNA barcode. The above methods and reagents can
prevent denaturation.
[0298] Reducing damage by using sealed ends of dsDNA. Another
method for reducing damage to bead-bound DNA barcodes, and for
reducing damage to partially synthesized DNA barcodes, is to use
double stranded DNA (dsDNA) and to seal the ends of this dsDNA by
way of 7-aza-dATP and dGTP.
[0299] Reducing damage by avoiding proteic solvents, avoiding
strong acids and basis, avoiding strong reducing agents and
oxidants. The type of chemistry that is compatible with the
presence of deoxyribonucleic acids (DNA), whether bead-bound DNA or
DNA that is not bead-bound, may require absence of proteic
solvents, avoiding strong acidic conditions, avoiding strong basis
such as t-butyl lithium, avoiding strong reducing agents such as
lithium aluminum hydride, avoiding reagents that react with DNA
bases, such as some alkyl halides, and avoiding some oxidants (see,
Luk and Sats (2014) DNA-Compatible Chemistry (Chapter 4) in A
Handbook for DNA-Encoded Chemistry, 1.sup.st ed. John Wiley and
Sons, Inc.).
[0300] As stated elsewhere, the term "DNA barcode" can refer to a
polynucleotide that identifies a chemical compound in its entirety
while, in contrast, "DNA barcode module" can refer to only one of
the monomers that make up the chemical compound.
[0301] Reducing damage to nucleic acids by using DNA-compatible
chemistry. Satz et al, disclose various chemistries that are
compatible with bead-bound nucleic acids (Satz et al (2015)
Bioconjugate Chemistry. 26:1623-1632; correction in Satz et al
(2016) Bioconjugate Chem. 27:2580-2580). Although the descriptions
in Satz et al, supra, concern chemical reactions that are performed
on DNA/chemical library member conjugates, the types of
DNA-compatible chemistries that are described are also relevant,
where the organic chemistry is to be performed on a bead that
contains bead-bound compounds and bead-bound DNA.
[0302] DNA-compatible reactions for the formation of benzimidazole
compounds, imidazolidinone compounds, quinazolinone compounds,
isoindolinone compunds, thiazole comopunds, and imidazopyridine
compounds are disclosed (see, Satz et al, Table 1, entries
1-6).
[0303] Moreover, DNA-compatible protecting groups are disclosed as
including, alloc deprotection, BOC deprotection, t-butyl ester
hydrolysis, methyl/ethyl ester hydrolysis, and nitro reduction with
hydrazine and Raney nickel (see, Satz et al, Table 1, entries
7-11).
[0304] Furthermore, methods for coupling reagents to DNA are
disclosed, where the coupling occurs with a functional group that
is already attached to the DNA. The methods include Suzuki
coupling, an optimized procedure for the Sonogashira coupling
between an alkyne and an arylhalide, the conversion of aldehydes to
alkynes using dimethyl-1-diazo-2-oxopropylphosphonate, a new method
for triazole cyclo addition directly from purified alkyne, an
improved method for reaction of isocyanate building blocks with an
amine functionalized DNA where the improved reaction occurs with
isocyanate reagent at pH 9.4 buffer (see, Satz, et al, Table 1,
entries 12-15).
[0305] Additional methods for coupling reagents to DNA are
disclosed, where the coupling occurs to a functional group already
attached to the DNA. These include a method where aprimary amine is
conjugated to DNA, an optimized procedure to form DNA-conjugated
thioureas, a method to alkylate secondary amines and the
bis-alkylation of aliphatic primary amindes, monoalkylation of a
primary amine DNA-conjugate, using hetarylhalides as building
blocks that can be reacted with amine-functionalized DNA-conjugate,
and methods for Wittig reactions (see, Satz et al, Table 1, entries
16-20).
[0306] Reducing damaged DNA by way of DNA repair enzymes. Various
proteins, including enzymes, DNA-damage binding proteins, and
helicases, are available for repairing DNA damage. What is
commercially available is DNA repair proteins that can repair
oxidative damage, radiation-induced damage, UV light-induced
damage, damage from formaldehyde adducts, and damage taking the
form of alkyl group adducts. Glycoside enzyme, which remove damaged
bases (but do not cleave ssDNA or dsDNA) are available to repair
5-formyluracil, deoxyuridine, and 5-hydroxymethyluracil. T4PDG is
available to repair pyrimidine dimers. hNEIL1 as well as Fpg are
available to repair oxidized pyrimidines, oxidized purines,
apurinic sites, and apyrimidinic sites. EndoVIII is available to
repair oxidized pyrimidine and apyrimidinic sites. EndoV is
available for repairing mismatches. HaaG is a glycosylase that is
available for repairing alkylated purines. Where a DNA repair
enzyme leaves a gap, where double stranded DNA has a gap where one
or more continuous deoxyribonucleotides are missing in one of the
strands, then various DNA polymerases are available for filling in
the gap (see, Catalog (2018) New England BioLabs, Ipswich,
Mass.).
[0307] A variety of DNA repair enzymes and DNA repair systems have
been isolated from mammals, yeast, and bacteria. These include
those that mediate nucleotide excision repair (NER), direct repair,
base excision repair, transcription-coupled DNA repair, and
recombinational repair. Interstrand DNA crosslinks can be repaired
by combined use of NER and homologous recombination. Direct repair
includes repair of cyclobutane pyrimidine dimers and 6-4 products,
by way of photolyase enzymes. Direct repair also includes removal
of O.sup.6-methyl from O.sup.6-methylguanine by DNA
methyltarnsferase. See, Sancar et al (2004) Ann. Rev. Biochem.
73:39-85; Hu, Sancar (2017) J. Biol. Chem. 292:15588-15597.
[0308] The present disclosure provides systems, reagents, and
methods for repairing damage to bead-bound DNA barcodes by treating
with a DNA repair enzyme, or by a complex of DNA repair proteins,
and the like.
[0309] Reducing damage via coupling DNA to beads via their 3'-end.
Certain chemical transformation may damage exposed 3'-hydroxyl
groups of nucleic acids. For instance Mitsunobu reactions allow the
conversion of primary and secondary alcohols to esters, phenyl
ethers, thioethers and various other compounds, which might render
exposed 3'-ends unreactive to subsequent processing steps, or cause
the now modified 3'-end to participate in further chemcial
reactions. In some embodiments, the DNA tags may be attached to
beads via their 3'-end, so only the 5'-end is exposed to
solution.
[0310] The reagents, systems, and methods of the present disclosure
encompass bead-bound nucleic acids, such as a bead-bound DNA or a
bead-bound DNA tags, where coupling to the bead involves the
3'-terminus (or the 3'-end) of the DNA. Where ssDNA that comprises
a DNA barcode is coupled by way of the 3'-end, of the ssDNA,
sequencing can be initiated by hybridizing only one sequencing
primer, where this sequencing primer hybridizes upstream of the
entire DNA barcode, and where this hybridizing is at or near the
bead-bound end of the coupled ssDNA. As an alternative to using
only one sequencing primer, a plurality of sequencing primers can
be used, where each sequencing primer hybridizes upstream to a
particular DNA barcode module. For example, if a given DNA barcode
contains five DNA barcode modules, and where the DNA is coupled to
a bead by way of its 3'-end, the DNA barcode can include five
different primer annealing sites, where each primer annealing site
is located just upstream, or immediately upstream, of a given DNA
barcode module.
[0311] Double stranded DNA (dsDNA) coupling embodiments. In other
embodiments, what is coupled to the bead is dsDNA, where the
3'-terminus of only one of the strands in the dsDNA are coupled to
the bead. In a 5'-coupling embodiment that involves dsDNA, what can
be coupled is dsDNA, where the 5'-terminus of only one of the
strands of the dsDNA is coupled to the bead.
[0312] (V) Coupling Chemical Compounds to Beads
[0313] The present disclosure provides: (1) Linkers to attach
chemical library member to a substrate, such as a bead; (2) Linkers
to attach nucleic acid barcode to a substrate, such as a bead; (3)
Cleavable linkers, for example, cleavable by UV light, cleavable by
an enzyme such as a protease; (4) Non-cleavable linkers; (5)
Bifunctional linkers; (6) Multi-functional linkers; and (7)
Plurality of beads used for linking. Avalailable, for example, is
4-hydroxymethyl benzoic acid (HMBA) linker,
4-hydroxymethylphenylacetic acid linker (see, Camperi, Marani,
Cascone (2005) Tetrahedron Letters. 46:1561-1564).
[0314] A "non-cleavable linker" may be characterized as a linker
that cannot be detectably cleaved by any reagent, condition, or
environment, that is used during the steps of a given organic
chemistry procedure. Alternatively, a "non-cleavable linker" may be
characterized as a linker that cannot be cleaved, except by a
reagent, condition, or environment that is unacceptably destructive
towards other reactants, products, or reagents of a given organic
chemistry procedure.
[0315] A bifunctional linker, or other multifunctional linker, can
take the form of a fork (fork used by humans for consuming food),
where the handle of the fork is attached to a bead, and where each
tine of the fork are linked to one of a variety of chemicals. For
example, one tine can be linked to a chemical library member.
Another tine can be linked to a DNA barcode. Yet another tine of
the fork can be linked to a metal ion.
[0316] Regarding use of a multiplicity of beads, the present
disclosure provides multiple-bead embodiments, such as: (1) A first
bead containing attached nucleic acid barcode linked to a second
bead, where the second bead contains attached chemical library
member; (2) A first bead containing an attached nucleic acid
barcode linked to a second bead, where the second bead contains an
attached chemical library member, and where a third bead is
attached (to one or both of the first bead and second bead), and
where the third bead contains a covalently attached reagent. The
attached reagent can be an enzyme, where the enzyme is used for
assaying activity of the attached chemical library member.
[0317] (VI) Coupling Monomers Together to Make a Compound
[0318] Exemplary chemical monomers. Amino acid derivatives suitable
for use as chemical monomers for the compositions and methods of
the present disclosure are shown in FIG. 4. The figure indicates a
source of the chemicals, for example, AnaSpec EGT Group, Fremont,
Calif.; Sigman-Aldrich, St. Louis, Mo.; Acros Organics (part of
ThermoFisher Scientific), or Combi-Blocks, San Diego, Calif.
[0319] Additional chemical monomers are shown in FIGS. 22-27. Each
of FIGS. 22-27 provides the structure, chemical name, and an
associated DNA module barcode. As disclosed on the figures,
compounds 1-6 (FIG. 22), the respective barcodes are ACGT, ACTC,
AGAC, AGCG, AGTA, and ATAT. For compounds 7-10 (FIG. 23), the
respective barcodes are, ATGA, CACG, CAGC, and CATA. For compounds
11-16 (FIG. 24), the respective barcodes are, CGAG, CGCT, CGTC,
CTAC, CTGT, and GACT. For compounds 17-21 (FIG. 25), the respective
barcodes are GAGA, GCAC, GCTG, GTAG, and GTCA. For compounds 22-26
(FIG. 26), the respective barcodes are GTGC, TAGT, TATC, TCAG, and
TCGC. And for compounds 27-30 (FIG. 27), the respective barcodes
are TCTA, TGAT, TGCA, and TGTG. These barcodes are only exemplary.
For any given library of compounds, a different collection of DNA
barcodes may be used to identify each of the chemical monomers that
are used to build the compounds in that library.
[0320] Coupling reactions. The following describes coupling
chemical monomers to the bead and to each other, that is, where a
first step is coupling the first chemical monomer directly to the
bead by way of a cleavable linker, and where subsequent chemical
monomers are then connected to each other, one by one. The
conditions disclosed below are DNA compatible.
[0321] This describes methods to make three amino acid compounds on
Tentagel.RTM. beads. The Fmoc protected resin (1 mg, Rapp polymer
GmbH, 10 um, TentaGel M-NH2, 0.23 mmol/g) modified with
Fmoc-Photo-Linker, 4-{4-[1-(9-Fluorenylmethyloxycarbonylamino)
ethyl]-2-methoxy-5-nitrophenoxy} butanoic acid) or another
appropriate linker with Fmoc protection was suspended inside each
well of a reactor plate (Merck Millipore Ltd, 0.45 um hydrophobic
PTFE) in DMA (150 uL). The solvent was removed by application of a
vacuum to the bottom of the plate with a Resprep VM-96 vacuum
manifold. The Fmoc protecting group was removed by suspending the
resin in 150 uL of a mixture of 5% piperazine, 2% DBU in DMF. The
plate was sealed with an Excel Scientific Alumna Seal and shaken at
40 C for 15 min. The solvent was removed by an applied vacuum and
the deprotection procedure repeated for 5 min. After filtration
each well was washed with 150 uL each of 2.times.DMA, 3.times.DCM,
1.times.DMA with a vacuum applied between each wash to remove the
solvent. Each well of resin was then acylated by the appropriate
amino acid by adding 150 uL of a pre-activated mixture of 60 mM
Fmoc-amino acid, 80 mM Oxyma, 200 mM DIC and 80 mM
2,4,6-trimethylpyridine that was allowed to sit for 2 min at room
temperature. The plate was again sealed and shaken for 1 hr at 40
degrees C. After filtration each well was washed with 150 uL each
of 2.times.DMA and 3.times.DCM. The beads in each well were
re-suspended in 150 ul of DCM and each well's contents combined
through pipetting into a single receptacle. The combined beads are
thoroughly mixed and redistributed into the plate through pipetting
equal amounts in the appropriate wells (1 mg/well). The solvent was
removed by an applied vacuum and each well was ready for the next
appropriate step. For each additional amino acid coupling, first
the Fmoc deprotection step is repeated followed by the coupling
step with the desired amino acid. If a split and pool is required,
the combining and redistribution method is repeated.
[0322] This describes a method for creating 3-mer amino acid by
split-pool method on beads. The Fmoc protected resin (1 mg, Rapp
polymere GmbH, 10 um, TentaGel M-NH.sub.2, 0.23 mmol/g) modified
with Fmoc-Photo-Linker, 4-{4-[1-(9-Fluorenylmethyloxycarbonylamino)
ethyl]-2-methoxy-5-nitrophenoxy} butanoic acid) or any other
appropriate linker was suspended inside each well of a reactor
plate (Merck Millipore Ltd, 0.45 um hydrophobic PTFE) in DMA (150
uL). The solvent was removed by application of a vacuum to the
bottom of the plate with a Resprep.RTM. VM-96 vacuum manifold. The
Fmoc protecting group was removed by suspending the resin in 150 uL
of a mixture of 5% piperazine, 2% DBU in DMF. The plate was sealed
with an Excel Scientific Alumna Seal and shaken at 40 C for 15 min.
The solvent was removed by an applied vacuum and the deprotection
procedure repeated for 5 min. After filtration each well was washed
with 150 uL each of 2.times.DMA, 3.times.DCM, 1.times.DMA with a
vacuum applied between each wash to remove the solvent. Each well
of resin was then acylated by the appropriate AA by adding 150 uL
of a pre-activated mixture of 60 mM Fmoc-amino acid, 80 mM Oxyma,
200 mM DIC and 80 mM 2,4,6-trimethylpyridine that was allowed to
sit for 2 min at room temperature. The plate was again sealed and
shaken for 1 hr at 40 C. After filtration each well was washed with
150 uL each of 2.times.DMA, 3.times.DCM, 1.times.DMA. For each
additional AA coupling, first the Fmoc deprotection step is
repeated followed by the coupling step with the desired AA. To
analyze each successive coupling, a 1 mg portion of beads was
suspended in 100 uL DMSO and exposed to full power of the 365 nm
LED for two hours. The resin is filtered off and the filtrate
injected onto an Agilent 1100 series LCMS equipped with a Agilent
Poroshell SB-C-18, 3.0.times.50 mm, 2.7 um column. A gradient of 5%
CH3CN in 0.1% TFA in water to 100 CH3CN in 0.1% TFA over 4 min at a
flow rate of 1.2 mL/min and monitored at 220 nm was ran.
[0323] Experiment to make non-amino acid pendant with lenalidomide
(Revlimid.RTM.) attached. This would be attached to the last amino
acid after deprotection. This was also done in a spin. Each well of
resin was acylated (after an Fmoc deprotection) with 150 uL of a 5
min preaged mixture of 40 mM chloro acetic acid, 40 mM Oxyma, 80 mM
DIC, and 40 mM TMP in DMA. The plate was sealed and shaken at 40 C
for 1 hr. Each well was washed with 150 uL each of 3.times.DMA,
3.times.DCM, and 2.times.DMA. The resin was then re-suspended in a
suspension of 100 mM K2CO3 and 100 mM Rev in DMA. The plate was
sealed and shaken for 3 hrs at rt. The resin was washed with 150 uL
each of 2.times.50/50 DMA/water, 3.times.DMA, 3.times.DCM, and
2.times.DMA.
[0324] Defining the degree of fidelity of synthesis of a chemical
compound that is attached to a given bead. This concerns the
completed chemical compound, where the chemical compound is a
member of a chemical library. Each chemical compound may be made,
in part, or in full, from chemical monomers. The following
characterizes chemical compounds that are attached to a given bead.
This given bead may be the product of split-and-pool based
synthesis of a library of chemical compounds, where each bead
possesses a unique chemical compound.
[0325] Members of a chemical library can be synthesized on a solid
support, such as on a bead, by way of solid phase synthesis. Solid
phase synthesis of chemicals with peptide bonds is charactized by
use of one the following two chemical groups. The first chemical
group is, N-alpha-9-fluorenyl-methyloxycarbonyl (Fmoc, base
labile). The second chemical group is, tert-butyloxycarbonyl (tBoc,
acid labile) (see, Vagner, Barany, Lam (1996) Proc. Natl. Acad.
Sci. 93:8194-8199). Fmoc and tBoc are protecting groups that can be
used to protect pepide substrates, where the Fmoc group or tBoc
group is attached to the alpha-amino group (Sigler, Fuller,
Verlander (1983) Biopolymers. 22:2157-2162).
[0326] Preferably, at least 99.5%, at least 99.0%, at least 95%, at
least 90%, at least 85%, or at least 80% of the member of the
chemical library bound to a given bead, following completed
synthesis, has exactly the same chemical structure. It is possible
that incomplete coupling that might occur at one or more steps in
the multi-step synthesis of the chemical library member. For this
reason, the compositions of the present disclosure may be
characterized and limited by one of the following limits or
ranges.
[0327] What is also provided by the present disclosure are methods
and reagents where at least 5%, at least 10%, at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, or at least
70%, or at least 80%, or at least 90%, or at least 95%, or at least
99%, of the members of the chemical library bound to a given bead
has, following completed synthesis, exactly the same chemical
structure (these numbers do take into account, and reflect, errors
that might occur during solid phase synthesis, for example, failure
of one growing compound to receive one of the chemical monomers.
Also, these numbers do take into account, and reflect, chemical
damage to any of the monomers that might occur during solid phase
synthesis).
[0328] In exclusionary embodiments, the present disclosure can
exclude any method or reagent that does not meet one of the above
cut-off values for "exactly the same structure."
[0329] In an alternate embodiment, two beads, 3 beads, 4 beads, 5
beads, about 5-10 beads, about 10-20 beads, about 20-40 beads,
about 40-80 beads, in a population of beads, contain the same and
identical chemical compound (without taking into account any errors
in incorporation of chemical monomers during solid phase synthesis,
and without taking into account any chemical damage that occurs to
a chemical monomer during organic synthesis).
[0330] Introduction to click chemistry. According to Jewett et al,
"Click reactions are defined . . . as those that . . . [are]
selective, high yielding, and having good reaction kinetics. A
subclass of click reactions whose components are inert to the
surrounding biological milieu is termed biorthogonal" (Jewett and
Bertozzi (2010) Chem. Soc. Rev. 39:1272-1279). "Click chemistry"
can be used for joining small units together with heteroatom links,
such as carbon-X-carbon. Click chemistry can be used alone, or in
conjunction with other types of chemical reactions, for the
synthesis of drugs or drug candidates. Click chemistry works well
with procedures used for combinatorial chemistry. Reactions in
click chemistry are characterized by high yields, by being
irreversible, by insensitivity to oxygen or water. Classes of
chemical reactions used in "click chemistry" include: (1)
Cycloaddition reactions, especially from the 1,3-dipolar family and
from hetero-Diels Alder reactions; (2) Nucleophilic ring-opening
reactions, as with strained heterocyclic molecules such as
epoxides, aziridines, and cyclic sulfates; (4) Carbonyl chemistyr
of the non-aldol type; and (5) Addition to carbon-carbon multiple
bonds, as with oxidation reactions and some Michael addition
reactions. Click chemistry reactions are distinguished by their
high thermodynamic driving force, often greater than 20 kcal/mol
while, in contrast, non-click chemistry reactions involve forming
bonds with only a modest thermodynamic driving force (Kolb and
Sharpless (2003) Drug Discovery Today. 8:1128-1137, Kolb, Finn,
Sharpless (2001) Angew. Chem. Int. Ed. 40:2004-2021).
[0331] Tetrazine and trans-cyclooctene (TCO). Tetrazine, such as,
1,2,4,5-tetrazine, can react with trans-cyclooctene (TCO) by way of
a Diels-Alder cyclo addition (Devaraj, Haun, Weissleder (2009)
Angew. Chem. Intl. 48:7013-7016).
[0332] Hartig-Buchwald amination. Hartwig-Buchwald amination
reactions can be used in the solid-phase synthesis of
pharmaceuticals. This amination reactions is used to synthesize
carbon-nitrogen bonds, where the reaction involves: aryl-halide
plus amine (R.sub.1-NH--R.sub.2), as catalyzed by palladium, to
produce an aryl product where the amine replaces the halide, and
where the nitrogen of the amino group is directly attached to the
aromatic ring. The end-result is a product involving a carbon (of
aryl group) to nitrogen (of amino group) bond. Stated another way,
the reaction converts arylhalides into the corresponding anilines.
Hartwig-Buchwald amination is compatible with a variety of amines,
and is well-suited for combinatorial chemistry (Zimmermann and
Brase (2007) J. Comb. Chem. 9:1114-1137).
[0333] Huisgen cycloadditions. Huisgen 1,3-dipolar cycloaddition
reactions involve alkynes and organic azides. Alkynes have the
structure, R--C.dbd.CH. Azides have the structure,
R--N.sup.+.dbd.N.dbd.N.sup.-. Copper catalysts accelerate the rate
of the Huisgen cycloaddition reaction. The Huisgen reaction
operates by way of "click chemistry" or "click reactions." Huisgen
reaction, when catalyzed by copper, can produce a 1,2,3-triazole
nucleus suitable for making small molecule drugs. Huisgen reaction
is compatible with the presence of amino acid side chains, at least
when in a protected form. Molecules made with a 1,2,3-triazole may
possess a bond that is similar to the amide bonds of polypeptides,
and thus these molecules can be a surrogate for the peptide bond
(Angell and Burgess (2007) Chem. Soc. Rev. 36:1674-1689).
[0334] Peptide nucleic acids (PNAs). The present disclosure
provides the methods of split and pool chemistry, combinatorial
chemistry, or solid phase chemistry, for synthesizing peptide
nucleic acids. Peptide nucleic acids are analogues of
oligonucleotides. They resist hydrolysis by nucleases. They can
bind strongly to their target RNA sequences. Uptake of peptide
nucleic acids into cells can be enhanced by "cell penetrating
peptides" (Turner, Ivanova, Gait (2005) Nucleic Acids Res.
33:6837-6849; Koppelhus (2008) Bioconjug. Chem. 19:1526-1534).
Peptide nucleic acids can be made by solid phase synthesis and by
combinatorial synthesis (see, Quijano, Bahal, Glazer (2017) Yale J.
Biology Medicine. 90:583-598; Domling (2006) Nucleosides
Nucleotides. 17:1667-1670).
[0335] The present disclosure encompasses bead-bound compounds,
where the compound takes the form of only one monomer. For example,
this bead-bound compound can take the form of lenalidomide, or it
can take the form of lenalidomide with an attached carboxylic acid
group, or a form of lenalidomide where the amino group has been
modified with a small chemical moiety that bears a carboxylic acid
group, or where the compound is a lenalidomide analog that is a
stereoisomer or an enantiomer of lenalidomide.
[0336] (VII) split and Pool Synthesis and Parallel Synthesis
[0337] This concerns use of the "split and pool" method for
synthesizing a library of chemical compounds, and the method where
the "split and "pool" method is used for the simultaneous synthesis
of bead-bound chemical compounds and bead-bound DNA barcodes. This
also describes splitting and pooling to make a mixed set of
compounds. At a later point, what is disclosed below is coupling of
a non-amino acid, as well as the preparations of beads that are
modified by polyethylene glycol (PEG).
[0338] The present disclosure provides split and pool synthesis for
generating chemical libraries. In one embodiment, this method
involves the steps: (a) Split beads into different containers; (b)
Add a different building block to each container. For example,
where three container are used, add and react Species A to the
first containing, Species B to the second container; and Species C
to the third container, where the species become covalently bound
to attachment sites on whatever bead is in the container; (c) Pool
all beads together in one container; (d) Split beads into three
containers, (e) Add a different building block to each container,
where Species A is added to the first container, Species B is added
to the second container, and Species C is added to the third
container, where the species become covalently bound to the first
species that had been previously attached (see, Stockwell (2000)
Trends Biotechnol. 18:449-455).
[0339] The split-and-pool synthesis of the present disclosure
includes, either before or after each chemical coupling step
(making the chemical library member), a DNA-barcode coupling step,
where this DNA barcode identifies the chemical that is being
coupled in that step.
[0340] In exclusionary embodiments, the present disclosure can
exclude methods and reagents where, for a given step of parallel
synthesis, a barcode is attached prior to attaching a chemical.
Conversely, the present disclosure can exclude methods and reagents
where, for a given step of parallel synthesis, a chemical is
attached prior to attaching a barcode.
[0341] One characteristics of a bead-bound chemical library that is
prepared by the split and pool method, is that each bead will have
only one type of compound attached to it. Where there is incomplete
coupling, for example, if for a given split and pool step, only
4,000 out of 5,000 attachment sites was successfully coupled with
the desired chemical species, then some heterogeneity will
occur.
[0342] Parallel synthesis. In a preferred embodiment of the present
disclosure, parallel synthesis can be used for organic synthesis of
a chemical compound and of the associated DNA barcode. In actual
practice, modification of a bead by one more chemical monomers and
modification of the same bead by one more DNA barcode modules, is
not strictly in parallel. In actual practice, the bead receives one
more chemical unit (chemical monomer) followed by receiving a DNA
barcode module that encodes that particular chemical unit. The term
"parallel" refers to the fact that, as the polymer of chemical
library monomers grows, the polymer of DNA barcode module also
grows. When all of the DNA barcode modules have been attached to
the bead, to form either a CONCATENATED structure or an ORTHOGONAL
structure, the full-length DNA barcode is called a "DNA barcode"
(and not merely a DNA barcode module).
[0343] Ratio of Number of Externally Attached DNA Barcode to Total
Number of Attached Chemical Library Member.
[0344] This concerns external surfaces and internal surfaces of a
bead. For a given bead that has externally attached DNA barcodes
(without regard to number of internally attached DNA barcodes) and
attached chemical library member (attached to both external surface
as well as to internal surfaces), the ratio of number of externally
attached DNA barcode number total attached chemical library member
number can be, for example, about 0.1:100, about 0.2:100, about
0.5:100, about 1.0:100, about 2:100, about 5:100, about 10:100,
about 20:100, about 30:100, about 40:100, about 50:100, about
60:100, about 70:100, about 80:100, about 90:100, about 1:1, about
100:150, about 100:200; about 100:400; about 100:600, and the like.
In exclusionary embodiments, the present disclosure can exclude any
bead, or any population of beads, that fits into one of the above
values.
[0345] Homogeneity of DNA Barcode for a Typical Bead; Homogeneity
of Chemical Library Member for a Typical Bead
[0346] The present disclosure provides, for any given bead (or for
any population of beads) a "chemical library homogeneity" that is
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 92%, at least 94%, at least 96%, at least 98%, at
least 99.5%, and the like.
[0347] In less stringent embodiment, the present disclosure
provides, for any given bead or, alternatively, for any given
population of beads, a "chemical library homogeneity that is at
least 10%, at least 20%, at least 30%, at least 40%, or at least
50%.
[0348] Similarly, the present disclosure provides the above cut-off
values for assessing homogeneity of a barcode, such as a DNA
barcode.
[0349] Homogeneity for DNA barcode and homogeneity for a chemical
library member may be defined, in terms, of percent of total
population that conforms to the exact sequence as planned and
desired by the methods section of a lab manual or notebook.
[0350] In exclusionary embodiments, the present disclosure can
exclude any reagent, composition, or method, that does not conform
with one or more of the above cut-off values.
[0351] Where one assesses homogeneity of a population of beads, one
needs to account homogeneity for the sum of bead #1, bead #2, bead
#3, bead #4, bead #5, bead #6, bead #7, and so on, for the
situation where homogeneity is desired throughout the entire
population of beads.
[0352] In exclusionary embodiments, the present disclosure can
exclude any bead, or any population of beads, where homogeneity of
DNA barcode is not at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 92%, at least 94%, at least 96%,
at least 98%, at least 99.5%, and the like. Also, in exclusionary
embodiments, the present disclosure can exclude any bead, or any
population of beads, where homogeneity of chemical library member
is not at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 92%, at least 94%, at least 96%, at least 98%,
at least 99.5%, and the like.
[0353] Ratio of Internally Attached DNA Barcodes to Externally
Attached DNA Barcodes
[0354] In some embodiments of the present disclosure, it might be
desired to manufacture and use beads where DNA barcodes are mainly
attached on the exterior surface. One reason to NOT make and use
beads with internal DNA barcodes, is the low permeation of DNA
oligomers to the interior spaces, and low permeation of DNA ligases
to interior spaces (ligases for connecting DNA modules to each
other to create the finished DNA barcode). And for sequencing
purposes, a reason to NOT make and use internal DNA barcodes, is
low permeation of enzymes needed to amplify DNA needed for eventual
sequencing of the barcode. Yet another reason NOT to make and use
beads with internal DNA barcodes is to fee up interior space for
attaching members of the chemical library.
[0355] The present disclosure provides beads bearing DNA barcodes,
where the ratio of internally attached DNA barcodes to externally
attached DNA barcodes is about 0.1:100, about 0.2:100, about
0.4:100, about 0.8:100, about 1:100, about 2:100, about 4:100,
about 8:100, about 10:100, about 20:100, about 40:100, about
50:100, about 60:100, about 70:100, about 80:100, about 90:100,
about 1:1, and so on.
[0356] Also, the present disclosure provides beads bearing DNA
barcodes, where the ratio of internally attached DNA barcodes to
externally attached DNA barcodes is under 0.1:100, under 0.2:100,
under 0.4:100, under 0.8:100, under 1:100, under 2:100, under
4:100, under 8:100, under 10:100, under 20:100, under 40:100, under
50:100, under 60:100, under 70:100, under 80:100, under 90:100,
under 1:1, and so on.
[0357] A population of beads, in an aqueous suspension, can be
contacted to a substrate, such as a microwell array, resulting in
beads entering and occupying the microwells. The ratio of the
number of beads in the suspension to the number of microwells in
the substrate can be adjusted, to arrive at a desired occupancy.
For example, if the suspension contains only one bead, then every
microwell that contains a bead will contain only one bead, where
the remaining microwells will not contain any bead. If the
suspension contains 20,000 beads and if the substrate contains
200,000 microwells, then at least 180,000 microwells will be
totally empty of beads, and where most of the microwells that
contain a bead will contain only one bead. A small percentage of
occupied microwells will contain two beads.
[0358] In value embodiments, the ratio of bead number in the
suspension to microwell number can be about 0.2:100, about 0.4:100,
about 0.6:100, about 0.8:100, about 1:100, about 2:100, about
4:100, about 6:100, about 8:100, about 10:100, about 20:100, about
30:100, about 40:100, about 50:100, about 60:100; 80:100, about
100:100 (same as 1:1), about 2:1, about 4:1, about 6:1, about 8:1,
about 10:1, and the like.
[0359] In exclusionary embodiments, the present disclosure can
exclude any method or system, that falls into one of the above
values or ranges.
[0360] In range embodiments, the ratio of bead number in the
suspension to microwell number can be about 0.2:100 to about
0.4:100, about 0.4:100 to about 0.6:100, about 0.6:100 to about
0.8:100, about 0.6:100 to about 1:100, about 1:100 to about 2:100,
about 2:100 to about 4:100, about 4:100 to about 6:100, about
0.6:100 to about 8:100, about 8:100 to about 10:100, about 10:100
to about 20:100, about 20:100 to about 30:100, about 30:100 to
about 40:100, about 40:100 to about 50:100, about 50:100 to about
60:100, about 60:100 to 80:100; about 80:100 to about 100:100 (same
as 1:1), about 100:100 (same as 1:1) to about 2:1, about 2:1 to
about 4:1, about 4:1 to about 6:1, about 6:1 to about 8:1, about
8:1 to about 10:1, and the like.
[0361] In exclusionary embodiments, the present disclosure can
exclude any method or system, that falls into one of the above
values or ranges.
[0362] (VIII) Fabricating Picowells
[0363] Combination of UV light, photomask, and photoresist for
manufacturing a picowell array plate. Plates that include many
microwells or picowells can be fabricated as follows for use in the
present disclosure. In brief, a sandwich of three layers is
assembled. The top layer is photoresist. The middle layer is a
glass wafer. The bottom layer is a photomask. The picowells will be
carved out of the photoresist by UV light. After the picowells are
carved out of the flat sheet of photoresist, the photoresist
resembles a typical metal pan that contains cups for baking
muffins, and where the cups in the pan that are used for holding
muffin batter have angled sides. The UV light acts as an "un-cross
linker" because it breaks down the photoresist's polymer. After UV
treatment, solvent is added to wash out the UV treated photoresist,
leaving clean-looking picowells.
[0364] Rotating at an angle to create angled walls. Picowells with
angled walls are created as follows. The photomask has many
apertures, where each aperture corresponds to the desired bottom
dimension of the picowell. The bottom dimensions can include a
circumference, diameter, and a shape, that is, a round shape. The
top dimension of the well is created by directing an angled UV
light towards the apertures in the photomask while rotating the
light source or rotating the stage that holds the sandwich
(photomask/glass wafer/photoresist sandwich). With rotation, the
light source is not at a 90 degree angle to the
photomask/wafer/photoresist sandwich, but instead is slightly
angled away from the 90 degree point, in order to carve out angled
walls in each picowell. The resulting picowell array plate that
contains many picowells can be used as is. Alternatively, this
picowell array plate can be used as a mold for the inexpensive
creating of many picowell array plates.
[0365] Han et al describes equipment and reagents for manufacturing
microwell plates where the microwells have angled walls (see, Han
et al (2002) J. Semiconductor Technology and Science. 2:268-272).
What is described is a UV source, a contact stage, a tilting stage,
and the SU-8 photoresist. Fabrication begins with a single side
polished silicon wafer. SU-8 photoresist is coated on the wafer at
about 0.10 to 0.15 mm thick. Then, the photoresist is soft baked on
a 65 degrees C. hot plate for 10 min and then on a 95 degrees C.
hot plate for 30 minutes. The resulting photoresist/wafer sandwich
is contacted with a UV mask using a contact stage. The term
"Inclined and rotated UV lithography" refers to a method for
manufacturing microwell array plates or picowell array plates,
where each well has an angled wall. Here, the floor of the well has
a smaller diameter and the top of the well (where the top edge of
the well meets the flat surface of the plate) has a wider diameter.
For exposure with UV light, a turntable is used and where the UV
light is inclined (Han et al, supra). The mask is contacted with
the photoresist where each of the apertures in the mask are
circular. FIG. 8 of Han et al, supra, provides a picture of the
direction of UV light, the UV mask, the photoresist structure, the
wafer substrate, and the turntable. Han et al describes how to
manufacture a truncated cone. A soft material such as PDMS
(polydimethylsiloxane) may be poured over the cone array and cured,
whereupon peeling the PDMS layer, conical wells are formed.
[0366] Creating a mold for use in mass-production of picowell array
plates. Where a picowell array plate has been manufactured, epoxy
can be poured over the plate resulting in filling all of the
picowells and connecting all of the filled picowells with a
platform of epoxy. Once the epoxy has solidified, the solid
platform with the attached array of picoprotuberances is removed
(the picoprotuberance being the reverse of the desired picowell).
The solid platform with picoprotuberances is a reusable molding
that can be used for the manufacture of many picowell array
plates.
[0367] The procedure for making replicates from the epoxy mold (or
a cone array mold made of any hard material is called, "hot
embossing." Briefly, a substrate material is heated to its glass
transition temperature or softening temperature, at which point the
mold with picoprotrubances is uniformly pressed against the
heat-softened material. The mold can be separated from the
substrate after the picoprotrubances are transferred as
pico-invaginations into the substrate material. This disclosure
preferably discloses pico-cones and picowells as the patterns of
the mold and substrate, respectively.
[0368] Hot embossing, epoxy masters, and photoresist such as the
SU-8 photoresist are described (see, Bohl et al (2005) J.
Micromechanics and Microengineering. 15:1125-1130, Jeon et al
(2011) Biomed Microdevices. 13:325-333; Liu, Song, Zong (2014) J.
Micromechanics and Microengineering. 24:article ID:035009; del
Campo and Greiner (2007) J. Micromechanics and Microengineering.
17:R81-R95).
[0369] Other microwell plate embodiments. Plastic microwell arrays
can be manufactured by way of a thermal forming using a silicon
mold, where the silicon mold possesses an array of microwells, for
example, an array of 800,000 microwells. A high degree of control
that results in tapered geometries and smooth sidewalls, and
submicron tolerances can be created with use of a non-pulsed dry
etch process. In contrast, methods that use a pulsed dry etch
process, such as the Bosch process, can result in rough sidewalls
and lack of control over lateral dimensions during etching.
[0370] Using non-pulsed dry etch process, plastic arrays are
fabricated by thermally forming plastic on a silicon master that is
created by a non-pulsed isotropic dry etch process using a chrome
mask. This process uses three gases, Ar, SF.sub.6, and
C.sub.4F.sub.8. The process is conducted at a RF power between 1200
to 2000 Watts and a bias of 150 Watts. Fine-tuning of the taper of
the silicon mold with production of smooth sidewalls can be
accomplished by varying the gas flow between the three gases. What
is varied is the ratio of SF6 to C4F8, where the result of changing
the ratio is, for example, a tapered wall of the mold (the silicon
pillar) that resides at an angle of 18 degrees (very slanted
walls), 9 degrees (slightly slanted walls), or 2 degrees (walls
almost perpendicular to substrate) (see, Perry, Henley, and Ramsey
(Oct. 26-30, 2014) Development of Plastic Microwell Arrays for
Improved Replication Fidelity. 18.sup.th Int. Conference on
Miniaturized Systems for Chemistry and Life Sciences. San Antonio,
Tex. (pages 1700-1703).
[0371] In embodiments, the present disclosure provides a substate,
an array, a grid, a microfluidic device, and the like, that
includes an array of microwells. In one embodiment, all of the
microwells have essentially the same volume. This volume can be
about 1 femtoliters, about 2, about 4, about 6, about 8, about 10,
about 20, about 40, about 60, about 80, about 100, about 200, about
400, about 600 about 800, or about 1,000 femtoliters.
[0372] Moreover, the volume can take the form of a range between
any of the above two adjacent values, such as, the range of about
40 femtoliters to about 60 femtoliters. Also, the volume can take
the form of a range between any of the above two values that are
not immediately adjacent to each other in the above list.
[0373] Furthermore, the volume can be about 1 picoliters, about 2,
about 4, about 6, about 8, about 10, about 20, about 40, about 60,
about 80, about 100, about 200, about 400, about 600 about 800, or
about 1,000, about 2,000, about 5,000, about 10,000, about 20,000,
about 50,000, about 100,000, about 200,000, about 500,000, or about
1,000,000 picoliters. Also, the volume can take the form of a range
between any of the above two values that are not immediately
adjacent to each other in the above list.
[0374] In exclusionary embodiments, the present disclosure can
exclude any substrate comprising microwells, or any array
comprising microwells, where the volume of each microwell is
definable by one of the above values, or is definable by a range of
any of the above two values that are adjacent to each other, or is
definable by a range of any of the above two values that are not
adjacent to each other in the list.
[0375] Spherical plug (also known as capping beads) on microwells.
The present disclosure provides a spherical plug, or alternatively,
a porous spherical plug, for each and every well, or substantially
every well of a picowell array. A goal of the plug is to keep
drugs, drug candidates, cellular contents, and metabolites, inside
of the well. The plug also helps isolate the contents of picowells
from each other. The spherical plug may need not be perfectly
spherical, as long as the goal of covering the top (or opening, or
mouth), of the picowell may be satisfied. The well can have a top
diameter and a bottom diameter. Diameter of spherical plug, prior
to capping a well, is about 10 micrometers, about 30, about 35,
about 40, about 45, about 50, about 55, about 70, about 90, about
120 or about 200 micrometers. The plugs may be added to cover the
picowells by simply flowing them over the picowell array.
Centrifugation, pressure, agitation or other methods may be used to
jam the beads to the tops (or mouths or openings) of the picowells
to ensure tight sealing. In some embodiments, solvents may be used
to modify the swelling and/or size of the capping beads. In some
embodiments, the capping beads may be loaded in a solvent that
renders the beads shrunken, and once replaced by assay buffer, or a
different solvent, the capping beads are restored to their
originial sizes, or swell, thereby sealing the picowells tightly.
In some embodiments temperature may be used to swell or shring the
capping beads to obtain better seals at the mouths of picowells.
Where needed, capping beads may be held in place, and prevented
from falling further into the picowell, by one of the steps in a
stepped picowell array.
[0376] The capping beads may be the same type of beads that carry
the compounds of this disclosure, or may be beads of a different
type. In some embodiments, the capping beads may actually be the
compound bearing beads themselves. The capping beads may serve as
passive caps, preventing or slowing diffusion of molcules out of
the picowells, or the beads may be active beads, where functional
moieties attached to the capping beads may be used to capture
reagents from the picowells. In some embodiments, porous capping
beads may passively trap metabolites released from cell-based
assays performed inside picowells. In some embodiments, capping
beads may non-specifically capture cellular materials such as
lipid, proteins, carbohydrates and nucleic acids. In some
embodiments, the capping beads may be functionalized with
antibodies to specifically capture proteins released from healthy,
diseased, lysed or fixed cells. In some embodiments, the capping
beads may be functionalized with DNA or RNA oligonucleotides that
specifically capture cellular nucleic acids. In some embodiments,
the DNA or RNA functionalized capping beads may be used to capture
microRNA released from cells within the capped picowells. In some
embodiments picowells contain two beads, a compound containing bead
inside the picowell, and a capping bead covering the mouth of the
picowells. In some embodiments, the capping beads are also the
compound-bearing beads. In some embodiments, the capping beads
capture materials released from the compound beads. In some
embodiments, the capping beads capture a sampling of the compounds
released from compound-beads. In some embodiments, the capping
beads capture DNA barcodes released from the compound-beads. In
some embodiments, the capping beads capture different types of
analytes released from within the picowells they cap.
[0377] Relative hardness of cap and of picowell. A preferred
equipment is a microwell plate, where each microwell includes, in
its bottom surface, many thousands of picowells. Ability of a cap
to seat properly or to seal each picowell can be a function of the
hardness of the plastic that makes up the picowell's aperture and
the picowell's inner walls, relative to the hardness of the
cap.
[0378] Hardness of a plastic can be defined in terms of a
"durometer" value. Hardness is defined and tested as a material's
resistance to indentation. The hardness of the spherical plug, and
the hardness of the wall of the microwell can be defined in terms
of its "durometer." The hardness can be, for example, about 45,
about 50, about 55, about 60, about 65, about 70, about 75, about
80, about 85, about 90, about 95, or about 100. In attributing any
of these durometer values to a plastic substance or other
substance, one must also state which scale is used. For example,
the scale can be ASTM D2240 type A scale, which is used for softer
materials, or the ASTM D2240 type D scale, which is used for harder
materials (see, Silicon Design Manual, 6.sup.th ed., Albright
Technologies, Inc., Leominster, Mass.).
[0379] Shapes of picowells. In some embodiments, the picowells may
be cylindrical picowells where the diameter of the cylinder is
roughly similar at the top and the bottom of the picowell. In some
embodiments, the picowells may have a slight taper, with the top of
the picowells slightly larger than the bottom of the picowells. In
some embodiments, the picowells may be conical picowells, with
angles off normal anywhere between 1 degree to 30 degrees. In some
embodiments, the picowells are stepped picowells, where the
picowells have discontinuous steps from the top diameter to the
bottom diameter (as opposed to conical picowells whose diameter
change smoothly from the top to the bottom). In some embodiments,
the stepped picowells have a broad cylinder near the opening of the
picowell and a narrower cylinder near the bottom of the picowells.
In some embodiments, the stepped picowells may have multiple
discontinuous steps from the top to the bottom. In some embodiments
of multi-stepped picowells, the diameter at every rung may be
larger than the diameter of the rung below it. In some embodiments
a small bead may be deposited at the bottom of the stepped
picowell, and a capping bead may be deposited at the topmost
opening of the stepped-picowell. In some embodiments picowells may
contain more than 2 beads.
[0380] Methods to make stepped-picowells. FIG. 29 disclosed stepped
picowell. The embodiment shown has three compartments and two
steps. Top compartment is widest and is configured for accepting
cap where most of the top compartment is occupied by the cap in the
situation where the picowell is capped. Middle compartment is
configured for being occupied mainly by, or solely by, reagents.
Reagents can include buffer, enzyme substrates, one or more salts,
and a preservative or stabilizer such as dithiothreitol, RNAse
inhibitor, glycerol, or DMSO. Lowest compartment is configured for
being occupied by bead, that is, a bead with coupled both a DNA
library and with releasable compounds. In addition to bearing DNA
barcode and releasable compounds, the same bead can also bear a
"response capture element." Capping beads may be held in place, and
prevented from falling further into the picowell, by one of the
steps in a stepped picowell. In FIG. 29, structure 1 is cap.,
structure 2 is bead., and structure 3 is top region, which is
situated immediately above first step. Structure 4 is middle
region, which can be used for placing assay reagents. Middle region
is immediately above second step. Assay reagents in middle region
can diffuse into lowest regtion. Structure 5 is lowest region,
which can be used for placing a bead and for placing one or more
cells.
[0381] Regarding the space of the lowest compartment that is taken
up by the bead (assuming that only one bead is present in
picowell), the diameter of the bead can be about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 95%, or about 98%, of the diameter of the lowest compartment
(assuming that the picowell is a circular well). If picowell is not
a circular well, the above values can refer to widest dimension of
the well. In exclusionary embodiments, the present disclosure can
exclude any system or bead that does not meet any of the above
parameters.
[0382] Further regarding space taken up by the bead (assuming that
only one bead is present in picowell), about 50% of bead is in
lowest compartment and about 50% of same bead is in middle
compartment, where these parameters can also be: about 55% lowest
and about 65% middle; about 60% lowest and about 40% middle; about
65% lowest and about 45% middle; about 70% lowest and about 30%
middle, about 75% lowest and about 25% middle, about 80% lowest and
about 20% middle, about 85% lowest and about 15% middle, about 90%
lowest and about 10% middle, about 95% lowest and about 5% middle,
and about 100% lowest. For making these calculations the space
taken up by bead assumes (hypothetically) that the bead is not
porous. In exclusionary embodiments, the present disclosure can
exclude any system or bead that does not meet any of the above
parameters.
[0383] As with conical and cylindrical picowell, using a molding
system is one preferred embodiment to create stepped picowells. For
this purpose, a mold containing arrays of multilayered pillars is
desired, whereupon stamping into a thermoplastic or other curable
polymer substrate, an impression of stepped picowells may be
formed. A layered pillar array with multiple steps, each step of a
different diameter (smaller as it goes up) may be formed by a
multilayer lithography process. Briefly, a first layer of
photoresist is exposed, via a first mask, to crosslink the first
layer of the micropillar array. A second layer of photoresist may
be deposited directly on the (previously exposed) first layer, and
a second photomask may be used to crosslink a second pattern in the
second photoresist later, and so on. At the end of the
multiple-layer patterning, the stack of resist may be developed to
wash away the uncrosslinked regions, leaving an array of
multilayered pillars. Detailed protocols for creating multilayered
pillar arrays may be found in Francisco Perdigones et al., (Jan. 8,
2011). Microsystem Technologies for Biomedical Applications,
Biomedical Engineering, Trends in Electronics Anthony N. Laskovski,
IntechOpen. Once an array of multilayered pillars array is created,
standard processes may be used to imprint stepped picowell arrays
using the mold.
[0384] Removing the capping beads. In many embodiments it is
advantageous to sample the capping beads to study reactions,
analytes or cellular response to the chemical perturbations within
picowells. In some embodiments, the capping beads may be dislodged
from the mouths of the picowells by inverting the picowell array
and using mechanical agitation. In some embodiments solvents may be
used to shrink the picowells, rendering them easier to dislodge
from the mouths of picowells. In some embodiments, liquids of
higher density than the capping beads may be added on top of the
picowell array, causing the capping beads to raise by buoyancy and
float atop the high-density medium.
[0385] In some embodiments, the capping beads may be crosslinked to
each other, converting the capping beads to a capping sheet that
can be peeled off the top of the picowell array. In some
embodiments, a crosslinking gel may be poured over the capped
picowells, where the crosslinking gel crosslinks to the capping
beads, and to themselves, causing the capping beads to be embedded
into a crosslinked sheet that can be peeled off.
[0386] Preserving relative locations of picowells, in the form of
the peeled-off layer. It should be appreciated that in such
embodiments as when the capping beads are enmeshed into a gel layer
that can be peeled off, the relative locations of capping beads
with respect to each other and with respect to the picowells are
preserved in the peeled-off layer. This allows direct connection
between picowells, assays in picowells, the beads in the picowells,
and any materials captured in the capping beads.
[0387] In some embodiments, fiducial markers may be used to orient
the relative features of the picowell arrays to the capping beads
in the peeled-off-layer.
[0388] Fiducial markers to enable registration and alignment of
picowells. Arranging picowells in irregular arrays allows easy
identification of shifts and drifts during imaging of the picowell
arrays. In some embodiments, the picowells are arranged in an
irregular order to facilitate detection of optical and mechanical
drifts during imaging. In some embodiments, the picowell arrays
contain fiducial markers to help identify shifts and drifts during
imaging. In some embodiments, the fiducial markers are easily
identifiable shapes, patterns or features that are interspersed
between the picowells of the picowell array. In some embodiments, a
small number of picowells may themselves be arranged in an easily
identifiable pattern to allow easy registration in case of optical
or mechanical drifts during imaging. In some embodiments, external
marker, such as fluorescent beads, may be drizzled on the picowell
array to provide fiducial patterns.
[0389] Cap-free mat embodiments. Cap-free mat embodiment, at least
in some forms or examples, can take the form of a "capless film."
Instead of sealing openings at the top of picowells, for example,
for preventing evaporation of any cell culture medium or enzyme
assay medium that may be in the picowell, sealing can be
accomplished by way of a mat. Preferably, the mat is sized to cover
all of the picowells in a given picowell array. Alternatively, the
mat can be sized to cover a predetermined fraction of the picowells
in the array. The mat can be secured to the top of the picowell
plate, covering picowells and also covering the generally planar
top surface of the picowell plate that resides in between the
picowells. Secure contact can be achieve by one or more of: (i)
Maintaining constant pressure, for example, by a hard rubber platen
that sits on top of and serves as a weight on top of the matt; (ii)
Using a mat that is connected to a weight, such as hard rubber
platen; (iii) A reversible chemical adhesive, that can be applied
to the entire mat (in the situation where the mat is not be be an
absorbant mat). Whre the mat is to be an absorbent mat, the mat
contains circular absorbent pads that are surrounded by the
reversible chemical adhesive. Here, the mat is contacted with the
picowell array and aligned so that the circular absorbent pads
cover only the openings of each picowell, and do not "spill out"
over the opening to contact the planar surface of the picowell
plate.
[0390] Membranes for use as mat for contacting substantially planar
surface of picowell plate, and for use in capless-sealing of
picowells, are available. Flat sheet membranes, such as Dow Film
Tex, GE Osmonics, Microdyn Nadir, Toray, TriSep, Synder, Novamem,
Evonik, and Aquaporin flatt sheet membreans are available from
Sterlitech Corp, Kent, Wash. These include membranes made of
polyamide-TFC, cellulose acetate, polyamide-urea-TFC, cellulose
acetate blend, polypiperazine-amide-TFC, PES, composite
polyamide-TFC, PES, PAN, PVDF, PSUH, RC, PESH, polyether ether
ketone, polyimide, and so on. Pore size in terms of molecular
weight cutoffs include, 150 Da, 200 Da, 300 Da, 500 Da, 900 Da, 600
Da, 1,000 Da, 2,000 Da, 3,000 Da, 5,000 Da, 10,000 Da, 50,000 Da,
20,000 Da, 30,000 Da 70,000 Da, 100,000 Da, 200,000 Da, 300,000 Da,
400,000 Da, 500,000 Da, 800,000 Da, 3500 Da, 0.005 micrometers,
0.030 micrometers, 0.05 micrometers, 0.10 micrometers, 0.20
micrometers, and so on. Regarding the system, compositions,
reagents, and methods of the present disclosure, these cutoff
values can allow selective collection of certain classes of
compounds with exclusion of other classes of compounds. For
example, some of the above membranes can allow small molecule
metabolites to pass through and be absorbed by an absorbable mat,
while excluding proteins and other macromolecules. Flat sheet
membranes that are impermeable to all molecules, including water,
metal ions, salts, metabolites, proteins, and nucleic acids, are
also available for use in the systems, compositions, and methods of
the present disclosure.
[0391] Reversible adhesion can be mediated by "molecular velcro,"
for example, metalloporphyrin containing polymers with
pyridine-containing polymers (Sievers, Namyslo, Lederle, Huber
(2018) eXPRESS Polymer Letters. 12:556-568). Other molecular velcro
adhesives involve, L-3,4-dihydroxyphenyl alanine, complementary
strands of ssDNA (one type of ssDNA covalently attached to flat
upper surface of picowell plate, and other type of ssDNA covalently
attached to mat), copolymers containing catechol side chains, and
so on (see, Sievers, et al, supra). Also, reversible adhesion can
be mediated by a gallium adhesive, where degree of adhesion can be
controlled by slight changes in temperature (Metin Sitti (May 18,
2016) Switch and Stick. The chemical element gallium could be used
as a new reversible adhesive that allows its adhesive effect to be
switched on and off with ease. Max-planck-Gesellschaft). Yet
another reversible adhesive is available from DSM-Niaga Technology,
Zwoll, The Netherlands.
[0392] Absorbent substances (non-specific absorbents; specific
absorbents). Absorbent substances, which can be incorporated into a
mat to provide absorbent characteristics include "molecule sieve"
beads, such as Sepharose.RTM., Sephadex.RTM., Agarose.RTM., as well
as ion exchange beads made of DEAE cellulose,
carboxymethylcellulose, phosphocellulose, or any combination of the
above, all combined into one absorbent mat. Absorbent ligands
include those that are used in high pressure liquid chromatography
(HPLC) (see, BioRad catalog, Hercules, Calif.). Specific absorbents
include response-capture elements, such as poly(dT), which can
capture mRNA by way of hybridizing with polyA tail. Also, response
capture elements include exon-targeting RNA probs, antibodies, and
aptamers. Each or any combination of these can be covalently
attached to mat, to create an absorbent mat, where contacting
absorbent mat to top surface of picowell enables capture of aqueous
assay medium or aqueous cell culture medium that might be inside
picowells.
[0393] (IX) Depositing Beads into Picowells
[0394] Plates with picowells can take the form of a 96-well plate
where each of these 96 wells contains many thousands of picowells.
Also, plates with picowells can take the form of a 24-well plate,
where each of these 24 wells contains many thousands of picowells.
For the 96-well plate, each well can be filled using 0.1-0.2 mL of
a suspension of beads in water or in an aqueous solution. For the
24-well plate, each well can be filled using about 0.5 mL of a
suspension of beads in water or in an aqueous solution. Suspension
can be added using an ordinary pipet with a disposable tip. The
number of beads that are in the suspension can be that resulting in
about one third of the picowells containing only one bead, about
one third of the picowells containing two beads, and about one
third of the beads containing either no beads or more than two
beads. Also, the number of beads in the suspension can be that
resulting in the situation where, of the wells that do contain one
or more beads, at least 70%, at least 80%, at least 85%, at least
90%, at least 95%, or at least 98% of these wells contain only one
bead.
[0395] After the beads have settled, any excess liquid can be
removed by touching a pipet tip to the wall of each well of the 96
well plate, or by touching a pipet tip to the wall of each well of
the 24 well plate, and drawing off the excess liquid.
[0396] Regarding assay reagents, where the picowells are to be used
for carrying out reactions, for example, DNA sequencing,
biochemical assays, or assays of cultured cells, assay reagents can
be added to the picowells that already contain settled beads.
Adding the assay reagents is with a pipette, as described above for
initial addition of the bead suspensions. After the assay reagents
have equilibrated with the solution that is already in each
picowell, any excess solution that is in each of the 96 wells of
the 96-well plate, or any excess solution that is in each of the 24
wells of the 24-well plate, can drawn off with a pipet tip that
touches the wall of each of the 96 wells of the 96-well plate, or
that touches the wall of each of the 24 wells of the 24-well
plate.
[0397] Flow-cell embodiment of picowell array. Picowell array may
be part of a flow-cell, where a fluidic chamber with an inlet and
an outlet are mounted on top of the picowell array. In such
embodiments, beads of this disclosure, cells, and other assay
materials may be flowed in from the inlet and out through the
outlet. Gravity or centrifugal force may be used to lodge the beads
into the picowells as they are flowed through the flowcell.
[0398] (X) Sequencing Bead-Bound Nucleic Acids in Picowells
[0399] Bead-bound nucleic acids can be sequenced while still
attached to beads. Alternatively, or in addition, bead-bound
nucleic acids can be sequenced following cleavage of the DNA
barcode from the bead.
[0400] Cleaving the DNA barcode from the bead before sequencing. In
some embodiments, the present disclosure can encompass a method
where bead-bound DNA barcode is cleaved from the bead, thereby
releasing the DNA barcode in a soluble form, prior to
amplification, or prior to sequencing, or prior to any type of
sequence identification technique such as hybridizing with a
nucleic acid probe.
[0401] Exclusionary embodiments. In embodiments, the present
disclosure can exclude any method, associated reagents, system,
compositions, or beads, where a bead-bound DNA barcode is cleaved
prior to amplification, or prior to sequencing, or prior to any
type of sequence identification technique such as hybridizing with
a nucleic acid probe. Also, the present disclosure can exclude any
method where a polynucleotide comprising a DNA barcode is cleaved,
or where a nucleic acid comprising only part of a DNA barcode is
cleaved, prior to amplification, prior to sequencing, or prior to
any type of sequence identification technique such as hybridizing
with a nucleic acid probe.
[0402] Polymerase chain reaction (PCR); Quantitative PCR (qPCR).
The PCR method, as well as the qPCR method, depend on the 3-step
method involving: (1) Denaturing the DNA template at a high
temperature, annealing primers at a reduced temperature, and
finally extending the primer by way of DNA synthesis, as catalyzed
by DNA polymerase (Gadkar and Filion (2014) Curr. Issues Mol. Biol.
16:1-6). qPCR is also called, "real time PCR" (Kralik and Ricchi
(2017) Frontiers Microbiology. 8 (9 pages).
[0403] Recent modifications or improvements in the PCR method and
qPCR method include, using helicase-dependent (HDA) amplification,
using an internal amplification control, using locked nucleic acids
(LNA), and using additives that bind to inhibitors (Gadkar and
Filion (2014) Curr. Issues Mol. Biol. 16:1-6). Locked nucleic acids
provide the advantage of recognizing and binding its target with
extreme precision.
[0404] qPCR allows the simultaneous amplification and
quantification of a targeted DNA molecule. The qPCR method compares
the number of amplification cycles required for the response
curvecs to reach a particular fluorescence threshold (Pabinger,
Rodiger, Kriegner (2014) Biomolecular Detection Quantification.
1:23-33). Refsland et al provide a concise account of apparently
typical conditions for conducting qPCR (Refsland, Stenglein, Harris
(2010) Nucleic Acids Res. 38:4274-4284).
[0405] Guidance is available for designing and validating PCR
primers, and on variables such annealing temperature (Ta), melting
temperature (Tm), temperature of elongation step, type of buffer
(Bustin and Huggett (2017) Biomolecular Detection Quantification.
14:19-28).
[0406] Rolling circle amplification (RCA). DNA can be amplified
while attached to a bead. DNA in amplified form is easier to
sequence that non-amplified DNA. In the rolling circle
amplification method, DNA tags (the DNA barcode) is made single
stranded. Once single stranded, a splint oligo is added to bridge
the ends of the tag DNA, and this is followed by extension and
ligation of the splint oligo. Using DNA polymerase (minus
5'.fwdarw.3'exonuclease activity) ensures a ligatable junction
after the DNA catalyzes extension of the splint oligo. The
circularized DNA can then be subjected to rolling circle
amplification by adding a strand-displacing DNA polymerase, such as
phi29 DNA polymerase. The ability to perform rolling circle
amplification (RCA) on the DNA barcode tag permits the use of
synthesis chemistries that may be damaging to DNA, as any surviving
DNA molecules can be thermally amplified to sufficient quantities
to be easily sequenced. DNA can be made single-stranded by
exonuclease digestion, nicking, and melting at high temperature, or
by treating with sodium hydroxide.
[0407] Further details of rolling circle amplification (RCA) are
revealed by the following steps that can be used for conducting
RCA.
[0408] Step One: Start with bead-bound ssDNA. If the bead-bound DNA
is initially in a double stranded from (dsDNA), the strand that is
not to be used for RCA can be prepared so that a residue of thymine
(T) is replaced, at or very close to the bead-attachment terminus,
with a residue of uracil (U). If the dsDNA is prepared in this way,
uracil-N glycosidase can be used to cleave the uracil residue,
thereby leaving an unstable sugar phosphate (as part of the DNA
backbone), where this unstable location can be cleaved by
nuclease-treatment (Ostrander et al (1992) Proc. Natl. Acad. Sci.
89:3419-3423).
[0409] Step Two: Add a "splint oligo" to the bead-bound ssDNA. The
splint oligo is designed so that it hybridizes to about 10-20 base
pairs at the end (the 5'-end) of the ssDNA that is covalently
coupled to the bead, and so that it also hybridizes to about 10-20
base pairs at the free end (the 3'-end) of the bead-bound ssDNA.
The splint oligo does not need to bring the bead-bound end of the
ssDNA in close proximity to the free end of the bead-bound ssDNA.
All that is needed is for the far ends of the bead-bound ssDNA
sequence be tethered together, in order to form a huge loop.
[0410] Step Three: Add sulfolobus DNA polymerase IV, so that this
polymerase uses the huge loop of ssDNA as a template, for creating
a complementary huge loop that is covalently attached at one end to
the splint oligo.
[0411] Step Four: Use DNA ligase to covalently close the
complementary huge loop, where the result is circular ssDNA. It is
this closed circle of ssDNA that does the "rolling," during
RCA.
[0412] Step Five: Add DNA polymerase that has a strand displacement
activity, and add dNTPs. The added DNA polymerase covalently
attaches dNTPs to the bead-bound ssDNA, and the distal terminus of
the bead-bound ssDNA is extended to create a complementary copy of
what is on the "rolling circle," and then further extended to
create yet another complementary copy of what is on the "rolling
circle," and even more extended to create still another
complementary copy of what is on the "rolling circle." During this
process of potentially infinite amplification, continued activity
of DNA polymerase is made possible by the strand displacement
activity of the DNA polymerase.
[0413] Optionally, the method of the present disclosure includes
real-time monitoring of rolling circle amplification (RCA) by way
of fluorescent molecular beacons (Nilsson, Gullberg, Raap (2002)
Nucleic Acids Res. 30:e66 (7 pages)). Reagents for RCA are
available from Sigma-Aldrich (St. Louis, Mo.), Sygnis TruePrime
Technology (TruePrime.RTM. RCA kit), Heidelberg, Germany, and GE
Healthcare (TempliPhi 500.RTM. amplification kit). Fluorophores and
quenchers are available from ThermoFisher Scientific (Carlsbad,
Calif.), Molecular Probes (Eugene, Oreg.), Cayman Chemical (Ann
Arbor, Mich.), and Sigma-Aldrich (St. Louis, Mo.).
[0414] Step Six. Use the ssDNA that was amplified by RCA as a
template for PCR amplification, where primers are added, where
thermostable DNA polymerase is added, and where the PCR products
are subsequently sequenced by Next Generation Sequencing.
[0415] In one aspect of the present disclosure, the RCA-amplified
ssDNA is cleaved from the bead prior to PCR amplification that
makes PCR products. In another aspect of the present disclosure,
the PCR amplification that makes PCR products can be made without
cleaving the RCA-amplified ssDNA from the bead.
[0416] As described by Baner et al, "Through the RCA reaction, a
strand can be generated that represents many tandem copies of the
complement to the circularized molecule" (Baner, Nilsson, Landegren
(1998) Nucleic Acids Res. 26:5073-5078). Bacillus subtilis phase
phi29 DNA polymerase is a suitable enzyme, because of its strand
displacement activity and high processivity. RCA is similarly
characterized by Li et al as, "In RCA, a circular template is
amplified isothermally by a DNA polymerase phi29 with . . . strand
displacement properties. The long single-stranded DNA products
contain thousands of sequence repeats: (Li and Zhong (2007) Anal.
Chem. 79:9030-9038).
[0417] Sequencing of DNA barcodes of the present disclosure can be,
without implying any limitation, with methods of Vander Horn U.S.
Pat. No. 8,632,975, which is incorporated herein by reference in
its entirety. Also, the DNA barcodes of the present disclosure can
be sequenced, for example, by methods that use
sequencing-by-synthesis, such as the Sanger sequencing method, or
by methods that use "Next Generation sequencing."
[0418] Illumina method for DNA sequencing. Illumina method for DNA
sequencing is as follows. DNA can be fragmented to a size range of
100-400 base pairs (bp) by sonication (Hughes, Magrini, Demeter
(2014) PLoS Genet. 10:e1004462). In the Illumina method, DNA
libraries are made, where fragments of DNA from a cell or from
cells are modified by DNA adaptors (attached to termini of the
fragments). The The reaction product takes the form of a sandwich,
where the DNA to be sequenced is in the center of the sandwich. The
reaction product takes the form: (first adaptor)-(DNA to be
sequenced)-(second adaptor). The adaptor-DNA-adaptor complex is
then associated with yet another adaptor, where this other adaptor
is covalently attached to a solid surface. The solid surface can be
a flat plate. The solid surface has a lawn of many adaptors that
stick out of the flat surface. The adaptor has a DNA sequence that
is complementary to one of the adaptors that is in the sandwich.
Actually, the lawn contains two type of adaptors, where one adaptor
binds (hybridizes) to one of the adaptors in the complex, and
non-covalently tethers the complex to the plate. These may be
called the, "first lawn-bound adaptor" and the "second lawn-bound
adaptor." The first task of DNA polymerase, is to create a daughter
strand, using the tethered (but non-covalently bound) DNA as a
template and, when DNA polymerization occurs, the daughter strand
is in a form that is covalently attached to the "first lawn-bound
adaptor." This covalent link was generated by the catalytic action
of DNA polymerase. After the daughter strand is completely
sythesized, the distal end (the end that sticks out into the
medium) contains a DNA sequence that is complementary to the second
adaptor in the above-named sandwich. This DNA sequence that is
complementary, allows the distal end of the newly synthesized
daughter DNA to bend over and to hybridize to the "second
lawn-bound adaptor." What has been described aboe, is how both
adaptors of the sandwich are used, and how both the "first
lawn-bound adaptor" and the "second lawn-bound adaptor" are
used.
[0419] A cycle of reactions is then performed many times, where the
result is a cluster of amplified versions of the original dsDNA.
Actually, the cluster takes the form of covalently attached
(tethered) ssDNA molecules, where all of these ssDNA molecules
correspond to only one of the strands of the original dsDNA (dsDNA
isolated from a living cell or tissue). This cluster of tethered
ssDNA molecules is called a "polony." The generation of the polony
is by a technique called, "bridge amplification." Finally, after
bridge amplification and the creation of polonies, the reverse
strands that are covalently attached to the solid surface are
cleaved from its tetherings, washed away, and discarded, leaving
only the forward strands.
[0420] Information on the Illumina.RTM. method is available from
Goodwin, McPherson, McCombie (2016) Nature Rev. Genetics.
17:333-351, Gierahn, Wadsworth, Hughes (2017) Nature Methods.
14:395-398, Shendure and Hanlee (2008) Nature Biotechnology.
26:1135-1145; Reuter, Spacek, Snyder (2015) Molecular Cell.
58:586-597; Illumina Sequencing by Synthesis (5 minute video on
YouTube).
[0421] Sequencing by oligonucleotide ligation and detection (SOLiD
sequencing). SOLiD measures fluorescence intensities from
dye-labeled molecules to determine the sequence of DNA fragments. A
library of DNA fragments is prepared from the sample to be
sequenced and used to prepare clonal bead populations (with only
one species of fragment on the surface of each magnetic bead). The
fragments attached to the beads are given a universal P1 adapter
sequence attached so that the starting sequence of every fragment
is both known and identical. PCR is conducted and the resulting PCR
products that are attached to the beads are then covalently bound
to a slide.
[0422] Then, primers hybridize to the P1 adapter sequence within
the library template. A set of four fluorescently labelled di-base
probes compete for ligation to the sequencing primer. Specificity
of the di-base probe is achieved by interrogating every 1st and 2nd
base in each ligation reaction. Multiple cycles of ligation,
detection and cleavage are performed with the number of cycles
determining the eventual read length. Following a series of
ligation cycles, the extension product is removed and the template
is reset with a primer complementary to the n-1 position for a
second round of ligation cycles (see, Wu et al (2010) Nature
Methods. 7:336-337).
[0423] pH-based DNA sequencing. pH-Based DNA sequencing is a system
and method where, base incorporations are determined by measuring
hydrogen ions that are generated as byproducts of
polymerase-catalyzed extension reactions. DNA templates each having
a primer and polymerase operably bound are loaded into reaction
chambers or microwells, after which repeated cycles of
deoxynucleoside triphosphate (dNTP) addition and washing are
carried out. The DNA template is templates are attached as clonal
populations to a solid support. With each such incorporation a
hydrogen ion is released, and collectively a population of
templates releasing hydrogen ions causing detectable changes to the
local pH of the reaction chamber (see, Pourmand (2006) Proc. Nat'l.
Acad. Sci.103:6466-6470). The present disclosure can exclude
pH-based DNA sequencing.
[0424] Regarding the concatenated DNA barcode, the entire
concatenated DNA barcode can be sequenced in one run (where
sequencing of the entire concatenated DNA barcode requires only one
sequencing primer). Alternatively, some or all of the DNA barcode
modules that make up the concatenated DNA barcode can be subjected
to individual sequencing (where each of the individually-sequenced
DNA barcode modules gets its own sequencing primer). Regarding
orthogonal DNA barcodes, each of the DNA barcode modules that make
up the orthogonal DNA barcode needs its own, dedicated sequencing
primer, because of the fact that each DNA barcode module is
attached to its own site on the bead.
[0425] Exclusionary embodiments. In embodiments, the present
disclosure can exclude any system, device, combination of devices,
and method, that involves microfluidics, aqueous droplets that
reside in an oil medium, and aqueous droplets that are created
where a first channel containing aqueous reagents is joined with a
second channel containing an oil to create aqueous droplets that
move through an oil medium through a third channel that starts at
the joining area. Microfluidics devices and reagents are described
(see, e.g., Brouzes, Medkova, Savenelli (2009) Proc. Natl. Acad.
Sci. 106:14195-14200; Guo, Rotem, Hayman (2012) Lab Chip.
12:2146-2155; Debs, Utharala, Balyasnikova (2012) Proc. Natl. Acad.
Sci. 109:11570-11575; Sciambi and Abate (2015) Lab Chip.
15:47-51).
[0426] In other exclusionary embodiments, what can be excluded is
any reagent, composition, nucleic acid, or bead, that comprises a
"DNA headpiece" or an reagent, composition, nucleic acid, or bead,
that is covalently attached to a "DNA headpiece." MacConnell,
Price, Paegel (2017) ACS Combinatorial Science. 19:181-192, provide
an example of a DNA headpiece, where beads are functionalized with
azido DNA headpiece moieties.
[0427] Additional exclusionary embodiments relating to sequencing
methods and sequencing reagents. In embodiments, the present
disclosure can exclude reagents, systems, or methods that do not
involve use of a "reversible terminator" in DNA sequencing. Also,
what can be excluded is any reagent, system, or method, that do not
include methoxy blocking group. Moreover, what can be excluded is
any reagent, system, or method, that involves DNA sequencing, but
where the DNA being sequenced is not covalently bound to a bead at
the time at the time that information on the order of
polynucleotides is being detected and collected. Furthermore, what
can be excluded is any reagent, system, or method that amplifies a
DNA template prior to conducting sequencing reactions, for example,
amplification by PCR technique or by rolling circle technique. In
embodiments, what can be excluded is any method of barcoding, for
example, nucleic acid barcoding, that is concatenated (all
information on synthesis of a member of the chemical library
residing on one single nucleic acid). In another aspect, what can
be exluced is any method of barcoding, for example, nucleic acid
barcoding, that is orthogonal (information on synthesis of a given
monomer of a chemical library being dispersed on a plurality of
attachment positions on the bead). In an exclusionary embodiment
relating to DNA ligase, the present disclosure can exclude any
reagent, system, or method, that uses DNA ligase for connecting
modules of a nucleic acid barcode.
[0428] Fluorophores, quenchers, and FRET-based assays. The present
disclosure provides fluorophores and quenchers for screening
members of a chemical library, or for characterizing an isolated
member of a chemical library. FRET is Forster resonance energy
transfer.
[0429] Assays can be performed on bead-bound chemical libraries.
Also, assays can be performed on free chemical library members
shortly after cleavage from a bead, that is, performed in the same
microwell as the bead or performed in the same vicinity of a
hydrogel matrix as the bead. Moreover, assays can be performed on a
soluble chemical library member that had never been attached to any
bead, or that had been cleaved from a bead and then purified.
[0430] Fluorophores suitable as reagents of the present disclosure
include Alexa 350, Alexa 568, Alexa 594, Alexa 633, A647, Alexa
680, fluorescein, Pacific Blue, coumarin, Alexa 430, Alexa 488,
Alexa 532, Alexa 546, Alexa 660, ATTO655, ATTO647n, Setau-665 (SETA
Biochemicals, Urbana, Ill.), Cy2, Cy3, Cy3.5, Cy5, Cy5.5,
tetramethylrhodamine (TMR), Texas red, tetrachlorofluorescein
(TET), hexachlorofluorescein (HEX), and Joe dye
(4'-5'-dichloro-2',7'-dimethoxy-6-carboxyfluorescein), SYBR green I
(absorb 497 nm, emit 520 nm), 6-carboxyfluorescein (6-FAM) (absorbs
492 nm, emits 518 nm), 5-carboxyfluorescein (5-FAM) (absorbs 492
nm, emits 518 nm), FITC, and rhodamine. Quenchers include TAMRA
quencher, black hole quencher-1 (BHQ1), and black hole quencher-2
(BHQ2), and DABCYL quencher. Please note, as disclosed elsewhere in
this patent document, that TAMRA can be a fluorophore and it can
also be a quencher.
[0431] Guidance is available on reagents for FRET-based assays,
where the FRET reagent includes a fluorophore and quencher (see,
Johansson (2006) Choosing reporter-quencher pairs for efficient
quenching. Methods Mol. Biol. 335:17-29). An example of a
FRET-based assays including measuring the activity of a signal
peptidase (SpsB) with the substrate, "SceD peptide." The FRET-pair
attached to the peptide was 4-(4-dimethylaminophenylazo)
5-((2-aminoethyl) amino)-1-nepthalenesulfonic acid (see, Rao et al
(2009) FEBS J. 276:3222-3234). Another example comes from assays of
HIV-1 protease, with the peptide substrate, KVSLNFPIL. The
donor/acceptor FRET pair was EDANS (donor) and DABCYL (acceptor).
EDANS fluorescence can be quenched by DABCYL by way of resonance
energy transfer to the nonfluorescent DABCYL (see, Meng et al
(2015) J. Biomolecular Screening. 20:606-615). Yet another example
comes from assays of botulinum toxin. Activity of SNAP-25 can be
measured by using the substrate, BoNT-A. For FRET-based assays, the
substrate had an N-terminally linked fluorescein-isothiocyanate
(FITC) and the C-terminally linked quencher was,
4-(4-dimethylaminophenyl) diazenylbenzoic acid (DABSYL). The
peptide substrate corresponded to amino acids 190-201 of SNAP-25
(see, Rasooly and Do (2008) Appl. Environ. Microbiol.
74:4309-4313).
[0432] The present disclosure provides for reagents, compositions,
and methods for screening a library of compounds in order to
discover and identify enzyme inhibitors, enzyme activators, and to
discover compounds that can enhance the rate of in vivo degradation
of a given protein. These reagents, compositions, and methods can
use FRET-based assays and, alternatively, they can use assays other
than FRET-based assays.
[0433] Molecular beacons are described (see, Baruch, Jefferey,
Bogyo (2004) Trends Cell Biology. 14:29-35). A molecular beacon is
a reagent where a fluorophore is bound, by way of a linker, to a
quencher. The linker may be cleavable by a nuclease, and thus
measure nuclease activity. The present disclosure provides for
methods to screen chemical libraries for identifying nuclease
inhibitors and, alternatively, for identifying nuclease activators.
Feng et al have described the use of molecular beacons and use of
FRET-based assays for measuring activity of various nucleases
(Feng, Duan, Liu (2009) Angew Chem. Int. Ed. Engl. 48:5316-5321).
Feng et al, showed use of FRET-based assays for measuring activity
of various restriction enzymes.
[0434] (XI) Releasing Bead-Bound Compounds
[0435] Cleavable linkers. What is provided is linkers that are not
cleavable. Also, what is provided are cleavable linkers (see,
Holmes and Jones ((1995) J. Org. Chem. 60:2318-2319; Whitehouse et
al (1997) Tetrahedron Lett. 38:7851-7852, and Yoo and Greenberg
((1995) J. Org. Chem. 60:3358-3364, as cited by Gordon et al (1999)
J. Chem. Technology Biotechnology. 74:835-851). Cleavable linkers
also include an acyl sulphonamide linkers that reside alkaline
hydrolysis, as well as activated N-alkyl derivatives which are
cleaved under mild conditions, and also traceless linkers based on
aryl-silicon bonds, and traceless linkers based on silyl ether
linkages (described on page 839 and 842 of Gordon et al (1999) J.
Chemical Technology Biotechnology. 74:835-851). Moreover, what is
provided is a linker based on tartaric acid which, upon cleavage,
generates a C-terminal aldehyde, where cleavage is by periodate
oxidation (see, Paulick et al (2006) J. Comb. Chem. 8:417-426).
[0436] FIGS. 3A-3I disclose various cleavable linkers that are
suitable for the compositions and methods of the present
disclosure. FIGS. 3A-3I are reproduced from Table 1 of: Yinliang
Yang (2014) Design of Cleavable Linkers and Applications in
Chemical Proteomics. Technische Universitat Munchen Lehrstuhl fur
Chemie der Biopolymere. From FIGS. 3A-3I, cleavable linkers that
are preferred for the present disclosure are linkers A, C, D, E, F,
G, and I. Linker E was used in the experimental results disclosed
herein. Cleavage conditions for these are DTT (linker A),
Na.sub.2SO.sub.4 (linker C), Na.sub.2SO.sub.4 (linker D), UV light
(linker E), UV light (linker F), UV light (linker G), and TEV
protease (linker I). These particular cleavage conditions are
gentle and are not expected to damage the bead, to damage the
bead-bound compound, or to damage any chemical library member (the
unit) of the bead-bound compound.
[0437] Chemically cleavable linkers that are compatible with
click-chemistry. Qian et al (2013) describes a number of cleavable
linkers that are compatible with click-chemistry (Qian, Martell,
Pace (2013) ChemBioChem. 14:1410-1414). These include linkers with
an azo bond, where the azo bond is cleavable with dithionite. This
linker has the following structure:
R.sub.1-benzene1-N.dbd.N-benzene2-R.sub.2. The first benzene ring
has a hydroxy group para to R.sub.1, and the second benzene ring
has a carbonyl group that links to R.sub.2, where this carbonyl
group is para to the azo moiety.
[0438] Photolabile cleavable linkers. The present disclosure
encompasses photocleavable linkers that have an o-nitrobenzyl
group. This group can be cleaved by irradiation at 330-370 nm (see,
Saran and Burke (2007) Bioconjugate Chem. 18:275-279; Mikkelsen,
Grier, Mortensen (2018) ACS Combinatorial Science. DOI:10.1021). A
linker with a shorter photolysis time than o-nitrobenzyl linker is
2-(2-nitrophenyl)-propyloxycarbonyl (NPPOC) linker. A variation of
o-nitrobenzyl linker is o-nitrobenzylamino linker. When attached to
a peptide chain, and when subsequently cleaved, this linker
releases an amide. Linker with an o-nitroveratryl group are
available, and these have shorter photolysis time and greater
release yields than unsubstituted o-nitrobenzyl linkers. Also
available are phenacyl linkers, benzoin linkers, and pivaloyl
linkers (see, Mikkelsen et al (2018) ACS Combinatorial Science.
DOI:10.1021).
[0439] Linkers with photocleavable ether bonds are available. This
photocleavable linker can be used where the linker is attached to a
bead and where the cleavable group is an "R group," and after
cleavage, the released group takes the form of ROH (see, Glatthar
and Giese (2000) Organic Letters. 2:2315-2317). Also available are
linkers with photocleavable ester bonds (see, Rich et al (1975)
97:1575; Renil and Pillai (1994) Tetrahedron Lett. 35:3809-3812;
Holmes (1997) J. Org. Chem. 62:2370-2380, as cited by Glatthar and
Giese, supra). Ether bonds in linkers can be cleaved by acid, base,
oxidation, reduction, and fluoride sensitive silyl-oxygen bond
cleavage, and photolysis (Glatthar and Giese, supra).
[0440] Another photocleavable linker, which has been used to link a
peptide (R.sub.1) and a nucleic acid (R.sub.2), is as follows.
R.sub.1 is connected directly to the methylene moiety of a benzyl
group. Para to the methylene group is a ring-attached nitro group.
Meta to the methylene moiety is a ring-attached ethyl group. The
1-carbon of the ethyl group bears a phosphate. To an oxygen atom of
this phosphate is attached the R.sub.2 group (Olejnik et al (1999)
Nucleic Acids Res. 27:4626-4631).
[0441] Akerblom et al, discloses photolabile linkers of the
alpha-methyl 2-nitrobenzyl type, containing amino, hydroxyl, bromo,
and methylamino groups, and also 4-nitrophenoxycarbonyl activated
hydroxyl and amino groups (see, Akerblom and Nyren (1997) Molecular
Diversity. 3:137-148). Cathepsin B can cleava a linker with the
target sequence, "valine-citrulline" (Dal Corso, Cazzamalli, Neri
(2017) Bioconjugate Chemistry. 28:1826-1833).
[0442] Enzyme-cleavable linkers. Linkers that are cleavable by
enzymes, such as proteases, are available (see, Leriche, Chisholm,
Wagner (2012) Bioorganic Medicinal Chem. 20:571-582). The
hydroxymethylphenoxy linker can be cleaved with chymotrypsin
(Maltman, Bejugam, Flitsch (2005) Organic Biomolecular Chem.
3:2505-2507). Linkers that are cleavable with tobacco etch virus
protease are available (see, Weerapana, Speers, Cravatt (2007)
Nature Protocols. 2:1414-1425; Dieterich, Link, Graumann (2006)
Proc. Nat'l. Acad. Sci. 103:9482-9487). The linker sequences LVPRG
and LVPRGS can be cleaved by thrombin (Jenny, Mann, Lundblad (2003)
Protein Expression Purification. 31:1-11). Plasmin-cleavable
linkers are available (Devy, Blacher, Noel (2004) FASEB J.
18:565-567).
[0443] Bead-bound release-monitor. The present disclosure provides
a novel and unique release-monitor that is capable of assessing
release of bead-bound compounds. The release-monitor takes the form
of a bead-bound complex of fluorophore and quencher, where the
fluorophore is connected to the bead by way of a cleavable linker.
Preferably, the cleavable linker is a photocleavable linker.
Preferably, the bead-bound release-monitor is situated in a
dedicated picowell, where that picowell does not contain any other
type of bead. With severing of the photocleavable linker, the
fluorophore is released from the bead, diffuses into the medium in
the picowell, achieves some distance from the bead-bound quencher,
where the result is an increase in fluorescence that is
proportional to the amount of release. The increase in fluorescence
allows the calculation of the concentration of the free fluorophore
that is in the picowell and, more importantly, allows calculation
of the amount of chemical compounds that are released from other
beads that are situated in other wells.
[0444] To summarize, the bead-bound release-monitor is situated in
its own dedicated well, where other wells contained bead-bound
compounds that are drug candidates.
[0445] FIG. 8 discloses a simplified version of a preferred and
non-limiting example of a bead-bound release-monitor. The
release-monitor takes the form of a quencher that is held in the
vicinity of a fluorophore, resulting in quenching of the
fluorophore. In embodiments, quenching is at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, at least 99.5%, at least 99.8%, at least 99.9%, at least
99.95%, and so on. In a picowell, one bead is dedicated to being a
release-monitor, while another bead or beads are used for attaching
a compound and for attaching DNA library. Exposure of all of the
beads in a picowell to UV light result in simultaneous cleavage of
fluorophore and of the compound. QSY7 is a preferred quencher. The
structure and CAS number for QSY7 is as follows (see below):
[0446] CAS name/number: Xanthylium,
9-[2-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]
carbonyl]-1-piperidinyl]sulfonyl]phenyl]-3,6-bis(methylphenylamino)-,
chloride 304014-12-8
[0447] The increase in fluorescence that results from separation of
the fluorophore from the quencher can be used to infer the
concentration in the picowell of the simultaneously released
compound. Also, the increase in fluorescence that results from
separation of the fluorophore from the quencher can be used to
infer the number of molecules (molecules taking the form of the
compound that was formerly a bead-bound compound) that reside in
free form in the picowell. In a more preferred embodiment, the
release-monitor comprises a quencher and a fluorophore, where
cleavage results in the release of the fluorophore (and not release
of the quencher). This embodiment provides lower background noise
than the following less preferred embodiment. In a less preferred
embodiment, cleavage results in the release of the quencher, where
the read-out takes the form of the increase in fluorescence from
bead-bound fluorophore.
[0448] The release-monitor provides the user with a measure of the
concentration of the soluble compound, following UV-induced release
of the compound from the bead. In a preferred embodiment, one type
of bead is dedicated to being a release-monitor. By "dedicated,"
what this means is that this bead does not also contain bead-bound
compound and does not also contain bead-bound DNA library.
[0449] As a general proposition, just because a compound has been
released from a bead by cleavage of a photosensitive linker, it
should not be inferred that the compound has become a soluble
compound. First of all, please note that just because a compound is
considered to be "hydrophobic" or is considered to be
"water-insoluble" does not mean that none of the molecules are
freely moving in the solvent. For example, even cholesterol has a
measurable solubility in water (see, Saad and Higuchi (1965) Water
Solubility of Cholesterol. J. Pharmaceutical Sciences.
54:1205-1206). Moreover, biochemical efficacy of a bead-bound
water-insoluble compound can be increased, by way of surfactants,
detergents, additives such as DMSO, or carriers such as human serum
albumin. Thus, the release-monitor can be used to assess overall
concentration of compounds of limited water-solubility or of no
water-solubility, under the condition where the picowell contains
one of the above agents or, alternatively, where the
water-insoluble compound is released in the vicinity of the plasma
membrane of a living cell that is cultured inside of the
picowell.
[0450] FIG. 9 discloses a simplified version of a preferred
embodiment of bead-bound release-monitor, while FIG. 10 discloses a
complete and detailed structure of this preferred embodiment of
bead-bound release-monitor.
[0451] FIGS. 30A-30F provide data demonstrating use of bead-release
monitor, where bead is in a picowell. The bead-bound fluorophore,
which is bound using a light-cleavable linker, was TAMRA
(excitation wavelength 530 nm; emission wavelength 570 nm). The
figure shows time-course of release of the fluorophore from the
bead. This shows operation of the bead-bound release monitor,
acquisition of fluorescent data at t=0 seconds, t=1 seconds, t=11
seconds, and t=71 seconds. FIGS. 30A-30F also include insets
showing blowups of the smaller figures, for two of the four smaller
figures. FIGS. 30A-30F were obtained from incubation of
cathepsin-D, which is an aspartyl protease, with "Peptide Q-Fluor
Substrate" and beads. Reagents were placed into wells at 4 degrees
C. Ultraviolet light at 365 nm was used to cleave the fluorophore
from the bead, thereby releasing the fluorophore and separating it
from the quencher. A goal of this assay was to assess the time
course of release taking place in a separate well, where the
separate well contained a different type of bead. The different
type of bead had the same light-cleavable linker, but where this
light-cleavable linker was attached to pepstatin-A. Release of
pepstatin-A can bind to and inhibit an aspartyl protease that is in
the same assay medium. This setup with bead-bound pepstatin-A and
the aspartyl protease can serve as a positive control.
[0452] UV exposed through 20.times. objective. Image was obtained
with Gain=5; Exposure was 400 ms. Excite at TAMRA at 530 nm. TAMRA
emits at 570 nm.
[0453] FIG. 35 discloses further details on enzymatic assays, where
bead-bound pepstatin-A is released, and where the released
pepstatin-A results in enzyme inhibition. 10 .mu.m TentaGel beads
displaying photocleavable Pepstatin-A (positive control) and a
covalent Cy5 label, were mixed with 10 .mu.m TentaGel beads
displaying photocleavable Fmoc-Valine (negative control) in PBST
buffer. This bead population was introduced into picowells, then
buffer exchanged into a protease inhibition assay, including
Cathepsin-D protease and Peptide Q-Fluor substrate
(.lamda..sub.ex=480 nm, .lamda..sub.em=525 nm). Wells were
encapsulated by air, and entire slide exposed to UV (365 nm, 77
J/cm.sup.2), cleaving the photolabile linker, releasing the
compound to reach approximately 13 .mu.M. The flowcell was
incubated (30 min, 37.degree. C.). Wells containing positive
control beads should inhibit peptide proteolysis by Cathepsin-D,
resulting in low fluorescence signal. Wells containing negative
control beads should not show any Cathepsin-D inhibition, and
should be similar in fluorescence intensity to empty wells.
[0454] Terminology for quencher and fluorophore can change, for a
given chemical, depending on what other chemicals occur in the
immediate vicinity. Although the TAMRA that is used in the
laboratory data of the bead-bound release monitor is a fluorophore,
in other contexts, TAMRA can be a quencher. TAMRA acts as a
quencher in TaqMan.RTM. probes that contain FAM and TAMRA.
[0455] Additional accounts of experimental setup and laboratory
data. The present disclosure provides data on controlled
5(6)-Carboxytetramethylrhodamine (TAMRA) concentrations in
phosphate buffer (10 mM phosphate, 154 mM sodium, pH 8.0) within
filled pico-wells, compartmentalized by air. Fluorescence images
captured (10 ms, 2 ms exposures) and well-area quantitated by mean
pixel intensity (n 100) to generate a concentration vs fluorescence
intensity calibration curve. The above data take the form of a
standard curve, showing fluorescence at various predetermined
concentrations of free TAMRA (2, 10, 30, 60, 100 mM TAMRA). This
standard curve was prepared under two different conditions, that
is, where the photographic image was taken with a 2 millisecond
exposure or with a 10 millisecond exposure. The experiment used for
preparing the standard curve was conducted in picowells, but there
were not any beads used in this experiment (just known amounts of
TAMRA). The photographic image is not shown in this patent
document, because the data merely take the form of a standard
curve, which may also be called a calibration curve.
[0456] The experimental setup included the following. For Scheme
X), TentaGel-Lys(PCL1-Tamra)-QSY7 bead structure. QSY7 (gray)
quenches the Tamra fluorophore (orange) while covalently attached
to bead through a photocleavable linker (purple). Irradiation from
UV (365 nm) provides quantitative release of compounds in situ
[0457] FIGS. 31A-31B discloses emission data resulting after
catalytic action of aspartyl protease on quencher-fluorophore
substrate. Greater fluorescence means that the enzyme is more
catalytically active. Lesser fluorescence means that the enzyme is
less catalytically active, that is, there the enzyme is more
inhibited by a free inhibitor, where the inhibitor was freed from a
bead, and where freedom was obtained by cleavage of light-cleavable
linker. Images were captured following UV release and Cathepsin-D
assay incubation (.lamda..sub.ex=480 nm, .lamda..sub.em=525 nm).
Wells containing positive control beads could be identified
spectrally by Cy5 fluorophore (.lamda..sub.ex=645 nm,
.lamda..sub.em=665 nm, orange false color). A section was analyzed
with a line-plot across open well volume, Wells containing negative
control beads elicit no Cathepsin-D inhibition. Assay volume within
wells containing positive control beads are dark, indicating strong
inhibition. Assay volume within empty wells is comparable to wells
containing negative control beads.
[0458] FIG. 32 illustrates the following procedure. Further
regarding Scheme X), Picowell substrate (46 pL per well) is
enclosed in a flowcell, wells wetted under vacuum, a suspension of
TentaGel-Lys(PCL1-TAMRA)-QSY7 beads are introduced, and air pulled
across flow-cell, compartmentalizing each well (top). Flowcell is
irradiated by a UV LED (.lamda..sub.mean 365 nm) with controlled
luminous flux, allowed to equilibrate (20 min), before fluorescence
microscopy images taken to quantitate released compound (TAMRA)
concentration (bottom) (FIG. 32). In detail, FIG. 32 shows drawings
of cross-section of picowell, illustrating the steps where
picowells wetted in a flowcell, the step where beads in a
suspension are introduced over the picowells, resulting in one bead
per picowell, the step of drawing air across flowcell in order to
reduce excessive dispersion solution and resulting in a meniscus
dropping below the surface of the planar top surface of the
picowell plate, the step of controlled UV exposure (365 nm),
resulting in release of some TAN/IRA, and the step of provoking
light emission from TAMRA with detecting fluorescent signal with
fluorescent microscopy (excite 531/40 nm) (emit 594/40 nm). The
notation, "slash 40" refers to the bandwidth, that is, it means
that cut-off filters confined the light to the range of: 531 nm
plus 20 nm and minus 20 nm, and to 594 nm, plus 20 nm and minus 20
nm (this slash notation can be used for excitation wavelengths and
also to emission wavelengths).
[0459] The present inventors acquired photographs showing the
following data (see, FIGS. 33A-33F). Fluorescence emission
(.lamda..sub.ex 531/40 nm, .lamda..sub.em 593/40) of fluorophore
(TAMRA) released from 10-.mu.m TentaGel-Lys(PCL1-TAMRA)-QSY7 beads
after UV LED (365 nm) exposure in pico-well flow cell. A) No
significant emission above background prior to UV exposure (0
J/cm.sup.2), owed to the FRET quenching effect of QSY7. TAMRA
release allowed to reach equilibrium (20 min) following UV
exposures of (B) 25 J/cm.sup.2, (C) 257 J/cm.sup.2, (D) 489
J/cm.sup.2, (E) 721 J/cm.sup.2, (F) 953 J/cm.sup.2 then imaged
using appropriate exposure times. Fluorescence emission was
measured within the volume surrounding each bead to measure TAMRA
concentration (FIGS. 33A-33F) The notation, "slash 40" refers to
the bandwidth, that is, it means that cut-off filters confined the
light to the range of: 531 nm plus 20 nm and minus 20 nm (this
slash notation can be used for excitation wavelengths and also to
emission wavelengths).
[0460] The following is an interpretation, by the present
inventors, of some of the fluorescence data from testing and use of
the bead-bound release monitor (see, FIG. 34) Concentration of
bead-released TAMRA inside pico-wells (45 pL) after UV exposure
(365 nm). Image analysis used mean pixel intensity of the solution
surrounding bead-filled wells (n.gtoreq.14), normalized to image
exposure time, then correlated to standard curve of known TAMRA
concentrations in pico wells. Error bars represent 1.sigma.,
calculated from RSD %. UV released compound concentrations were 1.1
.mu.M (RSD % 8.9), 54.3 .mu.M (RSD % 5.2), 142 .mu.M (RSD % 4.2),
174 .mu.M (RSD % 7.7), 197.3 .mu.M (RSD % 10.1) (FIG. 34)
[0461] (XII) Biochemical Assays for Compounds (Assays that are not
Cell-Based)
[0462] A variety of biochemical assays are possible using beads
within picowells.
[0463] Non-limiting examples include binding assays, enzymatic
assays, catalytic assays, fluorescence based assays, luminescence
based assays, scattering based assays, and so on. Examples are
elaborated below.
[0464] Biochemical assays that are sensitive to inhibitors of
proteases and peptidases. Where the goal is to detect and then
develop a drug that inhibits a protease, screening assay can use a
mixture of a particular protease or peptidase, a suitable cleavable
substrate, and a color-based assay or a fluorescence-based assay
that is sensitive to the degree of inhibition by candidate drug
compounds. For example, one reagent can be a bead-bound compound,
where the compound has not yet been tested for activity. Another
reagent can take the form of bead-bound pepstatin (an established
inhibitor of HIV-1 protease) (Hilton and Wolkowicz (2010) PLoS ONE.
5:e10940 (7 pages)). Yet another reagent can be a cleavable
substrate of HIV-1 protease, and where cleavage by the HIV-1
protease results in a change in color or a change in fluorescence.
Postive-screening drug candidates are identified where a particular
assay (in a given microwell) results in a difference in color (or a
difference in fluorescence). The cleavable substrate takes the form
of a susceptible peptide that is covalently bound to and flanked by
a quencher and a fluorescer. Before cleavage, the fluorophore does
not fluoresce, because of the nearby quencher, but after cleavage,
fluorescence materializes (see, Lood et al (2017) PLoS ONE.
12:e0173919 (11 pages); Ekici et al (2009) Biochemistry.
48:5753-5759; Carmona et al (2006) Nature Protocols. 1:1971-1976).
The reagents and methods of the present disclosure encompass the
above-disclosed technology.
[0465] Enzyme-based screening assay for compounds that inhibit
ubiquitin ligases, where the reagents include MDM2 (enzyme) and p53
(substrate). Applicants have conducted working tests based on the
following technology. MDM2 regulates the amount of p53 in the cell.
MDM2 is overexpressed in some cancers. MDM2 is an enzyme, as shown
by the statement that, "In vitro studies have shown that purified
MDM2 . . . is sufficient to ubiquitinate . . . p53" (Leslie et al
(2015) J. Biol. Chem. 290:12941-12950). Applicant's goal is to
discover inhibitors of MDM2, where these inhibitors are expected to
reduce ubiquitination of p53 and thus reduce subsequent degradation
of p53. In view of the expected increase in p53 in the cell, an
inhibitor with the above property is expected to be useful for
treating cancer.
[0466] Applicants used the following enzyme-based assay for
assessing the influence of lenalidomide on ubiquitination of p53,
as mediated by MDM2/HDM2. Applicants used reagents from the
following kit: MDM2/HDM2 Ubiquitin Ligase Kit--p53 Substrate
(Boston Biochem, Cambridge, Mass.). One of the reagents used in the
assay was a bead with a covalently bound antibody. The bead was
TentaGel.RTM. M NH.sub.2 (cat. no. M30102, Rapp Polymere GmbH,
Germany) and the antibody was anti-human p53 monoclonal antibody,
biosynthesized in a mouse. MDM2 is an E3 ligase that can use p53 as
a substrate, where MDM2 catalyzes ubiquitination of the p53.
[0467] Goal of activating p53 for reducing cancer. A relation
between MDM2, the transcription factor called, "p53," and
anti-cancer therapy is suggested by the following description. The
description is, "MDM2 is an E3 ubiquitin ligase that ubiquitinates
p53, targeting it for proteasomal degradation" (Ortiz, Lozano
(2018) Oncogene. 37:332-340). p53 has tumor-suppressing activity.
p53 activity can be inhibited by MDM2. According to Wu et al, MDM2
is a, "p53-binding protein" (see, Wu, Buckley, Chernov (2015) Cell
Death Disease. 6:e 2035). Where a compound prevents ubiquitination
of p53, for example, by blocking interactions between MDM2 and p53,
the compound might be expected to function as an anti-cancer
drug.
[0468] Goal of the screening assay. A purpose of the screening
assay is to discover compounds that influence ubiquitination of
p53, for example, compounds that stimulate p53 ubiquitination and
compounds that inhibit p53 ubiquitination. In detail, the purpose
is to discover compounds that are inhibiting or activating, where
their effect is via MDM-2 and either E1 ligase, E2 ligase, or E3
ligase. MDM2 means, "murine double minute." MDM2 has been called
an, "E3 ubiquitin ligase." When MDM2 occurs in the cell, evidence
suggests its activity in catalyzing the ubiquitination of p53
requires a number of other proteins, such as CUL4A, DDB1, and RoC1
(see, Banks, Gavrilova (2006) Cell Cycle. 5:1719-1729; Nag et al
(2004) Cancer Res. 64:8152-8155). Banks et al have described a
physical interaction involving p53 and MDM2 as, "L2DTL, PCNA and
DDB1/CUL4A complexes were found to physically interact with p53
tumor suppressor and its regulator MDM2/HDM2" (Banks, Gavrilova
(2006) Cell Cycle. 5:1719-1729). Nag et al have also described a
physical interaction involving p53 and MDM2 as, "Cul4A functions as
an E3 ligase and participates in the proteolysis of several
regulatory proteins through the ubiquitin-proteasome pathway. Here,
we show that Cul4A associates with MDM2 and p53" (Nag et al (2004)
Cancer Res. 64:8152-8155).
[0469] Desired read-out from the bead-based assay for modulators of
p53 ubiquitination. Where screening compounds results in a
positive-screening hit, that is, where there is more AF488
fluorescence, this means that an ACTIVATOR has been discovered. And
where screening compounds results in a positive-screening hit,
where there is a REDUCTION in fluorescence, this means that an
INHIBITOR has been discovered. A compound that inhibits
ubiquitination of p53, suggests that the compound can be used for
treating cancer. Also a compound that specifically inhibits
ubiquitination of p53, that is, where the compound does not inhibit
ubiquitination of other proteins, or where the compound inhibits
ubiquitination of other proteins with inhibition that is less
severe than for p53, also suggests that the compound can be used
for treating cancer.
[0470] Materials. Materials included E3 Ligase kit K-200B from
Boston Biochem. Boston Biochem catalog describes this kit as:
Mdm2/HDM2 Ubiquitin Ligase Kit--p53 Substrate. The following
concerns Mdm2, which is part of this kit. This kit does not include
cereblon. Lenalidomide and similar compounds can bind to either
cereblon or to Mdm2, where the end-result is activation of
ubiquitin ligase. Materials also included Diamond White Glass
microscope slides, 25 mm.times.75 mm (Globe Scientific, Paramus,
N.J.). Corning Stirrer/Hot Plate (settings from zero to ten) 698
Watts, Model PC-420. N-hydroxy-succinimide (NETS). Methyltetrazine
(mTET). AlexaFluor488 (AF488) (ThermoFisher Scientific). TentaGel
beads M NH.sub.2 (cat. No. M30102) (Rapp Polymere GmbH). Parafilm
(Sigma-Aldrich, St. Louis, Mo.). FIG. 8 shows the structure of
Alexa Fluor.RTM. 488. The structure of Alexa Fluor 488 (AF488) is
shown in Product Information for
AlexaFluor488-Nanogold-Streptavidin (Nanoprobes, Inc., Yaphank,
N.Y.).
[0471] (XIII) Cell-Based Assays for Chemical Compounds
[0472] Cell-based assays that are conducted in a picowell can use
human cells, non-human cells, human cancer cells, non-human cancer
cells, bacterial cells, cells of a parasite such as plasmodium
cells. Also, cell-based assays can be conducted with human cells or
non-human cells that are "killed but metabolically active," that
is, where their genome has been cross-linked to allow metabolism
but to prevent cell division (see, U.S. Pat. Publ. No. 2007/0207170
of Dubensky, which is incorporated herein by reference in its
entirety). Moreover, cell-based assays can be conducted on
apoptotic cells, necrotic cells, or on dead cells. Cell-based
assays with bacterial cells can be used to screen for antibiotics.
Human cells that are infected with a virus can be used to screen
for anti-viral agents. Combinations of cells are provided for
cell-based assays. For example, combinations of dendritic cells and
T cells are provided to screen for and identify compounds that
stimulate antigen presentation or, alternatively, that impair
antigen presentation.
[0473] Cell-based assays can be based on a primary culture of
cells, for example, as obtained from a biopsy of normal tissue, a
biopsy from a solid tumor, or from a hematological cancer, or from
a circulating solid tumor cells. Also, cell-based assays can be
based on cells that have been passaged one or more times.
[0474] Cell-based assays that are conducted in a picowell can use a
culture that contains only one cell, or that contains two cells,
three cells, four cells, five cells, or about 2 cells, about 3
cells, about 4 cells, about 5 cells, or a plurality of cells, or
less than 3 cells, less than 4 cells, less than 5 cells, and so
on.
[0475] Applicants have conducted working tests based on the
following technology. This describes cell-based assays for
screening compound for the exemplary embodiment where lenalidomide
(test compound) inhibits ubiquitin-mediated proteolysis of a
transcription factor. The transcription factors include Ikaros and
Aiolos.
[0476] The present disclosure provides a cell-based assay that
screens compounds on a bead-bound compounds, and where screening is
done with a plate bearing many picowells. The components of the
cell-based assay include, a picowell for holding a bead-bound
chemical library, where each bead has attached to it substantially
only one, uniform type of compound. The compounds are released by
way of a cleavable linker. Mammalian cells are cultured in the
picowell. The picowell also includes culture medium. The presently
disclosed non-limiting example with lenalidomide is a
proof-of-principle example that can be used for screening chemical
libraries in order to discover other compounds that modulate
ubiquitination of a given target protein.
[0477] Shorter description of a cell-based assay. Recombinant cells
are used as a reagent for detecting and screening for compounds
that induce proteolysis of green fluorescent protein (GFP), where
the read-out that identifies a positively screening compound is the
situation where green-colored cells become colorless cells, or
cells with reduced green color. Regarding the mechanism of this
cell-based assay, the mechanism of action of lenalidomide in
causing green-colored cells become colorless cells, or cells with a
reduced green color, is that the lenalidomide binds to a protein
called, "cereblon." In the cell, cereblon is part of a complex of
proteins called, "E3 ubiquitin ligase." Cereblon is the direct
target of the anti-cancer drugs, lenalidomide, thalidomide, and
pomalidomide. The normal and constitutive activity of E3 ubiquitin
ligase, and its relation to cereblon, has been described as,
"cereblon . . . promotes proteosomal degradation [of target
proteins] by engaging the . . . E3 ubiquitin ligase" (see, Akuffo
et al (2018) J. Biol. Chem. 293:6187-6200). In contrast to the
normal activity of E3 ubiquitin ligase, when a drug such as
lenalidomide, thalidomide, or pomalidomide is added, the result is
that the, "lenalidomide, thalidomide, and pomalidomide . . .
promote[s] the ubiquitination and degradation of . . . substrates
by an E3 ubiquitin ligase . . . each of these drugs induces
degradation of transcription factors, IKZF1 and IKZF3" (Kronke et
al (2015) Nature. 523:183-188).
[0478] Regarding terminology, cereblon has been described as being
part of a complex of proteins that is called, "E3 ligase" and also
called, "E3 ubiquitin ligase." Generally, cereblon by itself is not
called an "E3 ligase. The following excerpts reveal how the word
"cereblon" is used. According to Akuffo et al (2018) J. Biol. Chem.
293:6187-6200, "Upon binding to thalidomide . . . the E3 ligase
substrate receptor cereblon . . . promotes proteosomal destruction
[of the substrate] by engaging the DDB1-CUL4A-Roc1-RBX1 E3
ubiquitinligase." Consistently, Yang et al (2018) J. Biol. Chem.
293:10141-10157, discloses that, "Cereblon . . . functions as a
substrate receptor of the cullin-4 RING E3 ligase to mediate
protein [the substrate] ubiquitination." Zhu et al (2014) Blood.
124:536-545, state that, "Thalidomide binds CRBN [cereblon] to
alter the function of the E3 ubiquitin ligase complex . . .
composed of CRBN, DDB1, and CUL4." Lopez-Girona et al (2012)
Leukemia. 26:2326-2335, state that, "studies identified E3 ligase
protein cereblon (CRBN) as a direct molecular target . . . of
thalidomide . . . CRBN and . . . DDB1 form a functional E3 ligase
complex with Cul4A and Roc1."
[0479] To view the big picture of the cell-based assay devised and
used by the Applicants, the first step is that lenalidomide is
added to cells. The last step is that IKZF1 and IKZF3 are degraded.
Where IKZF1 occurs as a fusion protein with GFP, then the last step
is that the entire fusion protein is degraded by the proteasome.
Similarly, where IKZF3 occurs as a fusion protein with GFP, then
the final step is that this entire fusion protein gets degraded by
the proteasome. The result of GFP degradation is that the cell,
which was once green-fluorescing cell, is turned into a
non-fluorescing cell.
[0480] Longer description of a cell-based assay. This concerns
names of proteins of E3 ubiquitin ligase (a complex of proteins),
names of proteins that bind to this complex, and names of proteins
that are the target of this complex. For these names, the published
literature is not consistent. Sometimes it refers to the protein by
the name of the protein, and sometimes it refers to the protein
using the name of the gene that encodes the protein. For this
reason, the following account uses the protein name together with
the gene name, such as "cereblom" (name of protein" and "CRBN"
(name of gene). Also, "Ikaros" is the name of a protein, while the
gene's name is IKZF1. Also, "Aiolos" is the name of a protein,
IKZF3 is the name of the gene. "Cullin-ring finger ligase-4" is the
name of a protein, and the gene's name is CRL4. "Regulator of
cullin-1" is the name of a protein, and the gene's name is ROC1.
ROC1 is also known as, RBX1 (Jia and Sun (2009) Cell Division.
4:16. DOI:10.1186. "Cullin-4A" is the name of a protein and the
gene's name is CUL4A. See, Schafer, Ye, Chopra (2018) Ann. Rheum.
Dis. DOI:10.1136; Chen, Peng, Hu (2015) Scientific Reports.
5:10667; Matyskiela et al (2016) Nature. 535:252-257; Akuffo et al
(2018) J. Biol. Chem. 293:6187-6200).
[0481] E3 ubiquitin ligase catalyzes the transfer of a residue of
ubiquitin to a target protein, where the consequence is that the
target protein gets sent to the proteasome for degradation. The E3
ligase catalyzes attachment of ubiquitin to one or more lysine
residues of the target protein. Humans express about 617 different
E3 ubiquitin ligase enzymes (see, Shearer et al (2015) Molecular
Cancer Res. 13:1523-1532). E3 ubiquitin ligase is a complex of
these proteins: DNA damage binding protein-1 (DDB1); Cullin-4
(CUL4A or CUL4B); Regulator of Cullins-1 (RoC1); and RING
Box-domain protein (RBX1). As stated above, RoC1 is the same
protein as RBX1 (see, Jia and Sun (2009) Cell Division. 4:16.
DOI:10.1186). When cereblon (CRBN) joins the E3 ubiquitin ligase
complex, the resulting larger complex is called: CRL4.sup.CRBN
(Matyskiela et al (2016) Nature. 535:252-257). The term "CRL4"
means, "Cullin-4 RING Ligase" (Gandhi et al (2013) Brit. J.
Haematol. 164:233-244; Chamberlain et al (2014) Nature Struct. Mol.
Biol. 21:803-809). The above discrepancies in nomenclature need to
be taken into account when reading the literature of cereblon.
[0482] The following are longer versions of the short excerpts
disclosed above. Shown below is yet another form of nomenclature,
namely, the term: "CRL4.sup.cRBN E3 ubiquitin ligase." The longer
account more fully integrates the various names and cellular
events. "The relation between cereblon (CRBN) and E3 ubiquitin
ligase complex has been described as, "cereblon (CRBN) promotes
proteosomal degradation [of target protein] by engaging the
DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase" (Akuffo et al (2018) J.
Biol. Chem. 293:6187-6200). Regarding anti-cancer drugs,
"lenalidomide, thalidomide, and pomalidomide . . . promote the
ubiquitination and degradation of . . . substrates by an E3
ubiquitin ligase. These compounds bind CRBN, the substrate adaptor
for the CRL4.sup.CRBN E3 ubiquitin ligase . . . each of these drugs
induces degradation of . . . transcription factors, IKZF1 and
IKZF3" (Kronke et al (2015) Nature. 523:183-188).
[0483] This concerns cell-based assays where any given microwell,
nanowell, or picowell contains a bead where bead has covalently
linked compounds, where the compound is attached via a cleavable
linker, and where the well contains one or more cultured mammalian
cells. Responses to compounds and to drug candidates of the present
disclosure can be assessed by way of one or more biomarkers.
[0484] Biomarkers include diagnostic biomarkers, biomarkers that
predict if a given patient will respond (get better) to a given
drug, and biomarkers that predict if a given patient will
experience unacceptable toxicity to a given drug (Brody, T. (2016)
Clinical Trials: Study Design, Endpoints and Biomarkers, Drug
Safety, and FDA and ICH Guidelines, 2.sup.nd ed., Elsevier, San
Diego, Calif.). The present disclosure makes use of yet another
kind of biomarker, namely, a biomarker that monitors response of a
patient to a given drug, after drug therapy has been initiated. To
give an example, the following concerns the biomarker
"peroxiredoxin6 (PRDX6) and lung cancer. According to Hughes et al,
"PRDX6 levels in cell media from . . . cell lines increased . . .
after gefitinib treatment vs. vehicle . . . PRDX6 accumulation over
time correlated positively with gefitinib sensitivity. Serum PRDX6
levels . . . increased markedly during the first 24 hours of
treatment . . . changes in serum PRDX6 during the course of
gefitinib treatment . . . offers . . . advantages over
imaging-based strategies for monitoring response to anti-EGFR
agents." Please note comment that the biomarker has advantages over
a more direct measure of efficacy of response, namely, use of
"imaging" to detect decrease in tumor size and numbers (Hughes et
al (2018) Cancer Biomarkers. 22:333-344). Other biomarkers that
monitor response to anti-cancer drugs include CA125 for monitoring
response to platin therapy for ovarian cancer, and serum HSPB1 for
monitoring response to chemotherapy with ovarian cancer (see, Rohr
et al (2016) Anticancer Res. 36:1015-1022; Stope et al (2016)
Anticancer Res. 36:3321-3327).
[0485] Cytokine expression. Responses can be assessed by measuring
expressed cytokines, such as IL-2, IL-4, IL-6, IL-10, IFN-gamma,
and TNF-alpha. These particular cytokines can be simultaneously
measured using gold nanostructures bearing antibodies that
specifically recognize one of these cytokines, where detection
involves plasmon resonance (Spackova, Wrobel, Homola (2016)
Proceedings of the IEEE. 104:2380-2408; Oh et al (2014) ACS Nano.
8:2667-2676). Cytokines expressed by single cells, such as a single
T cell, can be measured by way of fluorescent antibodies, in a
device that includes microwells (Zhu, Stybayeva (2009) Anal. Chem.
81:8150-8156). The above methods are useful as reagents and methods
for the present disclosure.
[0486] In some embodiments, antibodies to cytokines may be attached
to the walls of the picowells, wherein any cytokines released, or
differentially released, from cells, as a function of drug exposure
can be captured by the antibodies bound to the walls of the
picowells. The captured cytokines may be identified by a second set
of labeled antibodies. In some embodiments, antibodies for
cytokines may be attached to capping beads. The capping beads may
then be embedded in a crosslinking hydrogel sheet that may be
peeled off and subjected to further analysis, for example, via
ELISA, mass spectrometer or other analytical techniques.
[0487] Apoptosis. Real-time data on apoptosis, and early events in
apoptosis of single cells can be measured with Surface-Enhanced
Raman Spectroscopy (SERS) and with Localized Surface Plasmon
Resonance (LSPR) (see, Stojanovic, Schasfoort (2016) Sensing
Bio-Sensing Res. 7:48-54; Loo, Lau, Kong (2017) Micromachines.
8:338. DOI:10.3390). Stajanovic, supra, detects release from cells
of cytochrome C, EpCam, and CD49e. Loo et al, supra, measures
release from cell of cytochrome C, where detection involves a DNA
aptamer (this DNA aptamer works like an antibody). Zhou et al
detect early apoptosis in single cells using SERS, where what is
measured is phosphatidyl serine on the cell membrane (see, Zhou,
Wang, Yuan (2016) Analyst. 141:4293-4298). In addition to
collecting data on apoptosis, SERS can be used for assessing drug
activity by collecting data on stages of mitosis, release of
metabolites, expression of a biomolecule bound to the plasma
membrane (see, Cialla-May et al (2017) Chem. Soc. Rev.
46:3945-3961). Plasmon resonance can measure protein denaturation
and DNA fragmentation that occurs in apoptosis (see, Kang, Austin,
El-Sayed (2014) ACS Nano. 8:4883-4892). Plasmon resonance (SERS)
can distinguish between cancer cells and normal cells, by measuring
the percentage of mitotic proteins in the alpha helix form versus
in beta sheet form (Panikkanvalappil, Hira, El-Sayed (2014) J. Am.
Chem. Soc. 136:159-15968). The above methods are suitable as
reagents and methods for the present disclosure.
[0488] Apoptosis can also be measured in cultured cells in a method
not using plasmonic resonance, but that instead uses
immunocytochemistry using anti-cleaved caspase-3 antibody (Shih et
al (2017) Mol. Cancer Ther. 16:1212-1223).
[0489] General information on cell-based assays. Cell-based assays
of the present disclosure can be used to test responses from human
cancer cells, cells from a solid tumor, cells from a hematological
cancer, human stem cells, human hepatocytes, a pathogenic
bacterium, an infectious bacterium, human cells infected with a
bacterium, human cells infected with a virus, and so on. The assays
can detect morphological response of the cell, such as migration,
as well as genetic responses and biochemical responses.
[0490] Assays of the present disclosure can be designed to detect
response of cells that are situated inside a microwell, or to
detect response of cells that are situated outside a microwell,
such as in a nutrient medium situated as a layer above the array of
microwells. Also, assays of the present disclosure can be designed
to detect responses of cells, where cells and beads are situated
within a medium, where cells are situated within a medium and beads
are above or below the medium, where cells are situated on top of a
medium and where beads are situated above or within or below the
medium.
[0491] The present disclosure provides a population of cells to a
microwell array. In embodiments, at least about 5%, at least about
10%, at least about 20%, at least about 40%, at least about 60%, at
least about 80%, at least about 90%, at least about 95%, or at
least about 100%, of the population of cells resides inside the
microwells (and not in any region situated above the microwells).
In embodiments, the proportion of cells that resides inside of the
wells, with the rest being situated in a layer of nutrient medium
residing above the array of wells, can be about 10%, about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90%, about 95%, about 100%, or in any range defined by two of
these numbers, such as the range of "about 60% to about 90%."
[0492] Matrix for cells. For assays of biological activity of
cells, and where cells are exposed to compounds released from
beads, or where cells are exposed to bead-bound compounds, suitable
matrices include those that include one or more of the following:
poly-D-lysine (PDL), poly-L-lysine (PLL), poly-L-ornithine (PLO),
vitronectin, osteopontin, collagen, peptides that contain RGD
sequence, polypeptides that contain RGD sequence, laminin,
laminin/fibronectin complex, laminin/entactin complex, and so on.
Suitable matrices also include products available from Corning,
Inc., such as, PuraMatrix.RTM. Peptide Hydrogel.RTM., Cell-Tak.RTM.
cell and tissue adhesive, Matrigel.RTM., and so on. See, Corning
Life Sciences (2015) Corning Cell Culture Surfaces, Tewksbury,
Mass. (20 pages), De Castro, Orive, Pedraz (2005) J. Microencapsul.
22:303-315. In exclusionary embodiments, the present disclosure can
exclude any composition or method that includes one of the above
matrices or one of the above polymers.
[0493] In embodiments, the present disclosure provides an array,
where individual microwells contain a bead, one or more cells, and
either a solution (without any matrix) or a matrix or a combined
solution and matrix. The matrix can be a hydrogel, polylysine,
vitronectin, MatriGel.RTM., and so on.
[0494] Activity of bead-bound compounds or of bead-released
compounds can be conducted. Assays to assess activity can include,
activating or inhibiting an enzyme, activating or inhibiting a
cell-signaling cascade or an individual cell-signaling protein,
binding to an antibody (or to a complementarty determining region
(CDR) of an antibody, to a variable region of an antibody),
inhibiting the binding of a ligand or substrate to an enzyme (or to
an antibody, or to a variable region of an antibody).
[0495] For the above assays, the readout can be determined with
fluorescence assays, for example, involving a fluorophore linked to
a quencher (F-Q). The linker can be designed to be cleavable by an
endoprotease, DNAse, RNAse, or phosopholipase (see, Stefflova,
Zheng (2007) Frontiers Bioscience. 12:4709-4721). The term
"molecular beacon" refers to this type of F-Q molecule, however,
"molecular probe" has also been used to refer to constructs where
separation of F and Q is induced by hybridization, as in TaqMang
assays (Tyagi and Kramer (1996) Nature Biotechnol. 14:303-308;
Tsourkas, Behlke, Bao (2003) Nucleic Acids Res. 15:1319-1330).
[0496] Transcriptional profiling in response to drug exposure. The
DNA barcodes of this disclosure may be modified to contain
response-capture elements, where the response capture elements
capture the response of cells to perturbations encoded by the
encoding portions of the barcode. In some embodiments, the DNA
barcodes may terminate in a poly-T section (multiple repeats of the
thymidne nucloetide), wher the poly-T sequence may be used to
capture poly-A terminated mRNA molecules released from lysed cells.
In some embodiments, the response-capture sequence may be
complementary to genes of interest, thereby capturing the
expression profile of desired genes via hybridization to the beads
of this embodiment. In some embodiments, picowells may contain a
single cell picowell whose transcriptional profile is captured on
the bead. In some other embodiments, a plurality of cells may be be
contanined in the picowell whose transcriptional profile is being
captured.
[0497] In one exemplary workflow, the following procedure may be
followed to cature transcriptional response of cells to drusg. (a)
Picowells designed to capture single cells per well are provided.
(b) A compound-laden, DNA barcoded bead is introduced into the
picowells, such that one bead is present per picowell. (c)
Compounds are released from the beads in each picowell by
appropriate methods (UV treatent for compounds attached via UV
cleavable linker, diffusion in case of beads soaked in compoiunds,
acide cleavable, base cleavable, temperature cleavable etc., as
appropriate for the beads of the embodiment). (d) The picowells may
be isolated from each other via a capping bead that retains
contents within the picowell or by other means such as an air
barrier or an oil barrier on top of the picowells. (e) The cells in
the picowells are allowed to incubate in the presence of the
compounds released from the beads for a duration. (f) After a
suitable amount of time, say 1 hr, 2 hrs, 5 hrs, 9 hrs, 12 hrs, 15
hrs, 18 hrs, one day, 3 days, one week, two weeks, one months, or
another appopriate time based on the assay, the cells are lysed by
a lysing method. The lysing methods may involve addition of
detergents, repeated cycles of freezing and thawing, heating,
addition of membrane disrupting peptides, mechanical agitation or
other suitable means. (g) once lysed, the contents of the cell are
exposed to the bead within the picowell, at which time the
response-capture elements on the beads of the picowell are enabled
to capture the response they are designe for. In some embodiments,
the response capture are poly-T sequences which capture the
complete mRNA profile of the cell (or cells) within each picowell.
In some embodiments, the response-capture elements are designed to
capture specific DNA or RNA seqences from the cell. In some
embodiments, the transcriptional response of the cell may be
captured as a function of dosage (or concentration) of
compounds.
EXAMPLES
Example 1. First Workflow
[0498] The present disclosure provides methods, including that
outlined below as "First Workflow" and as "Second Workflow."
The First Workflow includes the steps: (1) Generate DELB, (2) Beads
into picowells, (3) Load assay reagents into picowells, (4) Release
bead-bound compounds, (5) Measure assay readout, (6) Rank the assay
readout, and (7) Generate a new set of DELBs.
[0499] Generate DELB. First, create the DNA encoded library on
beads (DELB). Each bead contains a population of the exact, same
compound, though slight departures from this may occur where some
of the manufactured compounds had incomplete couplings or were
suffered chemical damage, such as inadvertent oxidation.
[0500] Beads into picowells. Then, deposit beads in picowells. In a
preferred embodiment, each picowell gets only one bead. Each
picowell can have a round upper edge, a round lower edge, a solid
circular bottom, an open top, and a wall. The wall's bottom is
defined by the round upper edge and by the round lower edge. In a
preferred embodiment, the wall is angled, where the diameter of the
round upper edge is greater than the diameter of the round lower
edge. In this way, the wall (viewed by itself) resembles a slice of
an inverted cone. The picowell array can be prepared, so that there
is a redundancy of beads. In other words, the array can be prepared
so that two of the beads, out of the many thousands of beads that
are placed into the picowells, contain exactly the same compound.
The redundancy can be, e.g., 2 beads, 3 beads, 4 beads, 5 beads, 10
beads, 20 beads, 40 beads, 60 beads, 80 beads, 100 beads, and so
on, or about 2, about 3, about 4, about 10, about 20, about 40,
about 60, about 80, about 100, about 200, about 500, about 1,000
beads, and so on, or more than 2, more than 5, more than 10, more
than 20, more than 40, more than 60, more than 80, more than 100,
more than 200, more than 500, more than 1,000 beads, and so on.
[0501] Load assay reagents into picowells. Introduce reagents into
each picowell that can be used to assess biochemical activity of
each bead-bound compound. The biochemical activity can take the
form of a binding activity, enzyme inhibition activity, enzyme
activation activity, activity of a living mammalian cell (where the
molecular target is not known), activity of a living mammalian cell
(where the molecular target is known), and so on. The reagent can
take the form of a FRET reagent plus an enzyme. The FRET reagent
can be a fluorophore linked by way of a protease substrate to a
quencher. The enzyme can be a substrate of that protease, which is
cleavable by the protease. The bead-bound compound is being tested
for ability to inhibit the protease.
[0502] After loading assay materials, each picowell can be capped
by a film, or many or all of the picowells can be capped by one
film, or many or all of the picowells can be capped by a film with
pimples where each pimple fits into a picowell, or or where each
picowell is fitted with a porous sphere. In embodiments, about 5%
of the volume about 10% of the volume, about 20% of the volume,
about 30% of the volume, or about 40% of the volume of the sphere
fits into the picowell (where the remainder is flush with the
surface or resides above the surface). In embodiments, about 5%,
about 10%, about 20%, about 40%, about 60%, about 80%, about 90%,
or about 100% of the pimple fits into the picowell.
[0503] Release bead-bound compounds. Perform a step that causes
release of the bead-bound compound. In embodiments, the step can
cause release of about 0.1%, about 0.2%, about 0.1%, about 0.2%,
about 2%, about 5%, about 10%, about 20%, about 40%, about 60%,
about 80%, about 99%, or about 100% of the compounds that are
attached to a given bead. Release can be effected by light, by a
chemical reagent, by an enzyme, by a shift in temperature, by any
combination thereof, and so on.
[0504] Release can take the form of: (i) Single release, (ii)
Multiple release, (iii) Continual release. Multiple release, for
example, can take the form of several emissions of ultraviolet
light, where each emission is sufficient to cleave about 10% of the
bead-bound compound that happens to be attached to the bead at the
start of that light emission. Continual release, for example, can
take the form of continual emission of light over the course of one
hour, resulting in a steadily increasing concentrations of free
compound. In this situation, the steadily increasing concentrations
of free compound (cleaved compound) may be for the purpose of
titrating the target of that compound. A titration experiment of
this kind can be used to assess potency of a given compound. To
provide non-limiting examples, with a single release method, a
period of light exposure is followed by a subsequent period where
readout is taken, and with a continual release method, light
exposure continues during some, most, or all of the period where
readout is taken.
[0505] In exclusionary embodiments, the present disclosure can
exclude any method, reagent, composition, or system that uses
single release, that uses multiple release, or that uses continual
release.
[0506] Measure assay readout. Detect the above-disclosed
biochemical activity, and the influence of the released compound on
that activity. This biochemical activity can take the form of
enzymatic activity, activity of a reporter gene, genetic activity
(e.g., rate of transcription or translation), binding activity
(e.g., antigen to antibody), cellular activity (e.g., change in
migration, change in cell-signaling pathway, change in morphology).
Activity can be detected by fluorescence, chromogenic activity,
luminescence, light microscopy, TaqMang assays, molecular beacons,
mass spectrometry, Raman spectroscopy, Localized Surface Plasmon
Resonance (LSPR), Surface Plasmon-Coupled Emission (SPCE),
Surface-Enhanced Raman Scattering (SERS), and so on. Detection can
be with methods that are totally remote, such as fluorescence
detection or light microscopy or, alternatively, by methods that
involve taking a sample from the picowell. In one embodiment, a
sample that contains a mixture of reactants and products can be
withdrawn for analysis by way of a spherical porous sponge that is
partially inserted into one of the picowells.
[0507] Rank the assay readout. In this step, assay readouts from a
plurality of different compounds (each type of compound associated
with one particular bead), are ranked in terms of their ability to
activate, inhibit, or in some way to modulate the biochemical
activity.
[0508] Generate a new set of DELBs. The steps that are described
above inform the user of various compounds that exhibit a
biochemical activity. The information may take the form of one
compound with maximal activity, with the rest having about half
maximal activity or less. Alternatively, the information may take
the form of several compounds having a similar maximal activity,
with the other compounds having about half maximal activity or
less. A new set of DELBs can be created as follows. One or more of
the highest-ranking compounds (the lead compounds) can be used as a
basis for manufacturing a new set of DELBs, based on one or more of
the following non-limiting strategies: (i) Replacing an aliphatic
chain with a homolog, such as replacing a propanol side chain with
a butanol side chain; (ii) Replacing an aliphatic chain with an
isomer, such as replacing a propanol side chain with an isopropanol
side chain; (iii) Replacing a peptide bond with an analog of a
peptide bond, such as with a bond that cannot be hydrolyzed by
peptidases; (iv) Replacing one type of charged group with another
type of charged group, such as replacing a phosphate group with a
phosphonate, sulfate, sulfonate, or carboxyl group.
Example Two. Second Workflow
[0509] The Second Workflow involves picowells that are sealed with
caps. The caps can take the form of spheres of slightly greater
diameter than the diameter of the picowells, where this diameter is
measured at the top rim of the picowell (not measured at the bottom
of the picowell). The cap can be made to fit snuggly into the top
of the picowell by subjecting the entire picowell plate to
mild-gravity centrifugation. In Second Workflow, the caps take the
form of beads that contain linkers, where each linker is linked to
a compound. The linkers are cleavable linkers, where cleavage
released the compounds and allows them to diffuse to the cells.
This type of cap is called an "active cap." The Second Workflow
includes the steps, (1) Generate DELB, (2) Load assay reagents into
picowells, (3) Cap picowells with DELB, (4) Release bead-bound
compounds from the bead that acts as a cap, (5) Measure assay
readout, (6) Determine sequence of the DNA barcode that is on the
bead; (7) Rank the assay readout, and (8) Generate a new set of
DELBs.
Example 3. Release Control
[0510] This concerns controlling and monitoring release of
bead-bound compounds. Applicants devised the following procedure
for synthesizing bead-bound release-monitor. See, FIG. 11 and the
following text.
[0511] FIG. 11 describes steps in the organic synthesis of the
above exemplary embodiment of a bead-bound release-monitor.
[0512] Step 1. Provide the Resin
[0513] TentaGel.RTM. resin (M30102, 10 .mu.m NH2, 0.23 mmol/g, 10
mg; MB160230, 160 .mu.m RAM, 0.46 mmol/g, 2 mg) was weighed into a
tube (1.5 mL Eppendorf) and swelled (400 .mu.L, DMA).
[0514] Resin was transferred into fritted spin-column (MoBiCol.RTM.
spin column, Fisher Scientific), solvent removed through filter by
vacuum, and pendent Fmoc was deprotected (5% Piperazine with 2% DBU
in DMA, 400 .mu.L; 2.times.10 min at 40.degree. C.). The MoBiCol
spin column has a 10 micrometer large frit and a luer-lock cap.
[0515] Resin was filtered over vacuum, and washed (2.times.DMA, 400
.mu.L; 3.times.DCM, 400 .mu.L; 1.times.DMA, 400 .mu.L).
[0516] Step 2. Couple Lysine Linker to Resin
[0517] A solution was prepared containing L-Fmoc-Lys(Mtt)-OH (21
.mu.moles, 6.6 eq.), DIEA (42 .mu.moles, 13.3 eq.), COMU (21
.mu.moles, 6.6 eq.) mixed in DMA (350 .mu.L), incubated (1 min,
RT), then added to dry resin inside the fitted spin-column,
vortexed, and incubated (15 min, 40.degree. C.) to amidate the free
amine. Resin was filtered by vacuum, and this reaction was
repeated, once.
[0518] Resin was filtered over vacuum, and washed (2.times.DMA, 400
.mu.L; 3.times.DCM, 400 .mu.L; 1.times.DMA, 400 .mu.L).
[0519] Step 3. Remove the Fmoc Protecting Group
[0520] The pendent Fmoc was deprotected (5% Piperazine with 2% DBU
in DMA, 400 .mu.L; 2.times.10 min at 40.degree. C.).
[0521] Resin was filtered over vacuum, and washed (2.times.DMA, 400
.mu.L; 3.times.DCM, 400 .mu.L; 1.times.DMA, 400 .mu.L).
[0522] Step 4. Couple the Quencher
[0523] A solution was prepared containing QSY7-NHS (4.9 .mu.moles,
1.55 eq.), Oxyma (9.5 eq, 3.3 eq.), DIC (21 .mu.moles, 6.6 eq.),
TMP (3.5 .mu.moles, 1.1 eq.) mixed in DMA (350 incubated (1 min,
RT), then added to dry resin inside the fitted spin-column,
vortexed, and incubated (14 hr, 40.degree. C.) to amidate the free
amine.
[0524] Resin was filtered over vacuum, and washed (2.times.DMA, 400
.mu.L; 3.times.DCM, 400 .mu.L; 1.times.DMA, 400 .mu.L).
[0525] A solution was prepared containing Acetic Anhydride (80
.mu.moles, 25.3 eq.), TMP (80 .mu.moles, 25.3 eq.), mixed in DMA
(400 .mu.L), mixed then added to dry resin inside the fitted
spin-column, vortexed, and incubated (20 min, RT)
[0526] Resin was filtered over vacuum, washed (2.times.DMA, 400
.mu.L; 3.times.DCM), and incubated in DCM (1 hr, RT), then filtered
over vacuum and dried in vacuum chamber (30 min, 2.5 PSI)
[0527] Step 5. Remove the Mtt Protecting Group
[0528] Mtt deprotection cocktail was prepared containing TFA (96
.mu.L), Methanol (16 .mu.L), mixed in DCM (1488 .mu.L) giving
6:1:93% of TFA:Methanol:DCM solution.
[0529] Mtt deprotection cocktail was added to the fully dried resin
(400 .mu.L), mixed, eluted by filtration over vacuum, then
sequential aliquots of Mtt deprotection cocktail (4.times.400
.mu.L) were added, mixed, incubated (5 min, RT), and eluted for a
combined total incubation time of 20 min at RT.
[0530] Resin was filtered over vacuum, and washed (3.times.DCM, 400
.mu.L; 1.times.DMA, 400 .mu.L; 1.times.DMA with 2% DIEA, 400 .mu.L;
3.times.DMA, 400 .mu.L).
[0531] Step 6. Couple the Photocleavable Linker to Epsilon-Amino of
Lysine
[0532] A solution was prepared containing Fmoc-PCL-OH (32
.mu.moles, 10 eq.), Oxyma (32 .mu.moles, 10 eq.), DIC (50
.mu.moles, 15.8 eq.), TMP (32 .mu.moles, 10 eq.) mixed in DMA (400
.mu.L), incubated (1 min, RT), then added to dry resin inside the
fitted spin-column, vortexed, and incubated (14 hr, 40.degree. C.)
to amidate the free .epsilon.-amine.
[0533] Resin was filtered over vacuum, and washed (2.times.DMA, 400
.mu.L; 3.times.DCM, 400 .mu.L; lx DMA, 400 .mu.L).
[0534] Step 7. Remove the Fmoc Protecting Group from the Previously
Coupled Photocleavable Linker
[0535] The pendent Fmoc was deprotected (5% Piperazine with 2% DBU
in DMA, 400 .mu.L; 2.times.10 min at 40.degree. C.).
[0536] Resin was filtered over vacuum, and washed (2.times.DMA, 400
.mu.L; 3.times.DCM, 400 .mu.L; lx DMA, 400 .mu.L).
[0537] Step 8. Couple the Fluorophore
[0538] A solution was prepared containing TAMRA (6 .mu.moles, 1.9
eq.), TMP (24 .mu.moles, 7.6 eq.), COMU (16 .mu.moles, 5 eq.),
mixed in DMA (400 .mu.L), incubated (1 min, RT), then added to dry
resin inside the fritted spin-column, vortexed, and incubated with
mixing (2 hr, 40.degree. C., 800 RPM) to amidate the free
amine.
[0539] Resin was filtered over vacuum, and washed (2.times.DMA, 400
.mu.L; 3.times.DCM, 400 .mu.L; 2.times. DMA, 400 .mu.L;
2.times.DMSO), then incubated with mixing in DMSO (16 hr,
40.degree. C.).
[0540] The following provides a broader account of the
above-disclosed laboratory procedures.
[0541] Bi-functional linker attached to bead. Bi-functional linker
was synthesized in solution and attached to an amine-functionalized
beads. FIG. 11 discloses pathway of organic synthesis, starting
with lysine. Lysine-Boc was than connected by TCO linker. The main
part of the linker was took the form of polyethylene glycol (PEG)
with a nitrogen at one end. Boc was a leaving group in this
connecting reaction. The TCA that was used was actually a racemate
of hydroxy-TCO. The hydroxyl group of this TCO derivative was
connected to a carbon atom located four carbon atoms away from one
side of the double bond (this is the same thing as being located
three carbon atoms away from the other side of the double bond). As
shown in FIG. 11, the first product in the multi-step synthesis
took the form of Boc-lysine-linker-TCO. The hydroxyl group that was
once part of hydroxy-TCO is still attached to the TCO group, where
it is situated in between the aminated-polyethylene glycol group
and the TCO group (FIG. 11).
[0542] The second set in the synthetic pathway involved treatment
with HCl and addition of a photocleavable linker (PCL). The product
of this second step was the same as the product of the first step,
except with the Boc group replaced with the photocleavable linker.
The lysine moiety takes a central position in the product of the
second step. Regarding the lysine moiety, this lysine moiety has a
free carboxyl group, and in the third step of the procedure, an
aminated bead is connected to this free hydroxyl group, resulting
in the synthesis of a bead-bound reagent, where the reagent takes
the form of two branches, and where at the end of one branch is a
TCO tag, and where at the end of the other branch is an aromatic
ring bearing a cleavable bond. To attached a chemical monomer to
the distal end of the photocleavable linker, first the Fmoc group
is removed, and here the Fmoc group is replaced with a hydrogen
atom.
[0543] Removing Fmoc. According to Isidro-Llobet et al, "Fmoc . . .
is removed by bases mainly secondary amines, because they are
better at capturing the dibenzofulvene generated during the
removal" (Isidro-Llobet et al (2009) Chem. Rev. 109:2455-2504).
Alternatively, Fmoc can be removed by catalytic hydrogenolysis with
Pd/BaSO.sub.4, or by liquid ammonia and morpholine or
piperidine.
[0544] Removal of Fmoc group followed by attaching a chemical
monomer. Applicants then condensed a chemical monomer having a
carboxylic acid group, where the result was generation of an amide
bond. (This step not shown in any figure.)
Example 4. Cereblon-Based Assay for Active Compounds
[0545] Results from cell-based assays of compounds (cereblon-based
assay). Reagents and methods for cell-based assay. Applicants used
CCL-2 HeLa cells obtained from ATCC (American Type Culture
Collection, Manasses, Va.). Cell medium was Gibco DMEM high glucose
medium buffered with HEPES. Atmosphere above cell culture was
atmospheric air supplemented with 5% carbon dioxide, with the
incubator at 37 degrees C. Cell medium was DMEM plus 10% fetal
bovine serum, supplemented with GlutaMAX.RTM. (Gibco Thermofisher),
and also supplemented with non-essential amino acids and penicillin
plus streptomycin (Gibco Thermofisher, Waltham, Mass.). HeLa cells
were transfected with a construct taking the form of
LTR-CTCF-Promoter-IKZF1 (or IKZF3)-mNeon-P2A-mScar-LTR-CTCF.
mScarlet is an element used as a positive control. mScarlet encodes
red fluorescent protein called, "mScarlet" (see, Bindels et al
(2017) Nature Methods. 14:53-56). The promoter is doxycycline
indudicble promoter, which enables rapid onset induction and
titration of the substrate. P2A is an element situated in between
two other polypeptides. P2A functions, during translation, to
product two separate polypeptides, thus allowing the mScar
polypeptide to function as a positive control that produces red
light, without being influenced by ubiquitination and degradation
of the fusion protein consisting of IKZF1/Green Fluorescent Protein
(GFP). mNeonGreen is derived from the lancelet Branchiostoma
lanceolatum multimeric yellow fluorescence protein (Allele
Biotechnology, San Diego, Calif.). P2A is a region that allows
self-cleaving at a point in the P2A protein. More accurately, the
P2A peptide causes ribosomes to skip the synthesis of the
glycyl-prolyl peptide bond at the C-terminus of a 2A peptide,
leading to the cleavage between a 2A peptide and its immediate
downstream peptide (Kim, Lee, Li, Choi (2011) PLoS ONE. 6:e18556 (8
pages).
[0546] Demonstration of efficacy of cell-based assay for test
compounds. The following demonstrates use of a cell-based assay for
test compounds taking the form of lenalidomide and analogus of
lenalidomide. FIGS. 5A-51I disclose results from HeLa cells that
were transfected with lentiviral vector, where the vector expressed
Green Fluorescent Protein (GFP) and a red fluorescent protein
(mScarlet). Increasing the concentration of added lenalidomide
resulted in progressively less green fluorescence, and elimination
of green fluorescence at highest concentrations. But lenalidomide
did not substantially decrease red fluorescence. Top: Expression of
IKZF1/GFP fusion protein. Bottom: Expression of mScarlett control.
Lenalidomide was added at zero, 0.1, 1.0, or 10 micromolar.
[0547] FIGS. 6A-6H disclose results from HeLa cells that were
transfected with lentiviral vector, where the vector expressed
Green Fluorescent Protein (GFP) and red fluorescent protein
(mScarlet). Increasing concentration of added lenalidomide resulted
in progressively less green fluorescence, and elimination of green
fluorescence at highest concentrations. But lenalidomide did not
substantially decrease red fluorescence. Top: Expression of
IKZF3/GFP fusion protein. Bottom: Expression of mScarlett control.
Lenalidomide was added at zero, 0.1, 1.0, or 10 micromolar.
[0548] To summarize the pathway where lenalidomide causes
proteolysis of the fusion proteins, first lenalidomide is added to
the HeLa cells. Then, the lenalidomide binds to the cereblon that
naturally occurs in these cells. This cereblon occurs in a complex
with E3 ubiquitin ligase. E3 ubiquitin ligase responds to the
lenalidomide by tagging the recombinant IKZF1 fusion protein (or
the recombinant IKZF3 fusion protein) with ubiquitin. The
end-result is that the ubiquitin-tagged fusion protein is degraded
in the cell's proteasome.
[0549] Coating the picowell plates. This describes solutions that
are applied to the top surface of a picowell plate, but that do not
necessarily enter and coat inside of picowells. This is also about
solutions that are applied to the top surface of a picowell plate
and that enter the picowells, and that coat the bottom surface of
the picowells. Applicants added a solution of Pluronic.RTM. 127
(Sigma Aldrich, St. Louis, Mo.) to dry plastic. The result is a
surface that is hydrophilic, and no longer hydrophobic. Then, the
surface was washed with water. Then, phosphate buffered saline
(PBS) was added, where this PBS enters inside the picowells. Moving
air is applied by way of a vacuum, where the result is that it
causes small bubbles in the picowells to expand, and where the
bubbles are then replaced with the PBS, and where the end result is
that much of the picowell gets filled with PBS. Then, PBS was
replaced with vitronectin coating solution (AF-VMB-220) (PeproTech,
Rocky Hill, N.J.). Pluronics.RTM. 127 is:
H(OCH.sub.2CH.sub.2).sub.x(OCH.sub.2CHCH.sub.3).sub.y(OCH.sub.2CH.sub.2).-
sub.zOH. After applying the vitronectin coating solution,
Applicants incubated for 30 min at 37 degrees C. to allow the
coating solution to get into picowells. The Pluronic 127 coats the
ridges that separate the picowells, and the vitronectin is at
bottom of picowells. HeLa cells attach to vitronectin and when they
attach to the vitronectin, they adhere to the bottom of the
picowell.
[0550] HeLa cells were screened for successfully transfected cells
by way of flow cytometry. Two criteria were used simultaneously for
determining successful transfection. First, lenalidomide was added
to cell media 2 days before sorting by flow cytometry. A positive
cell was that which was red-plus and green-minus, where red-PLUS
meant that the cells were transfected with the gene encoding mScar,
and where green-MINUS meant that the lenalidomide had in fact
promoted the ubiquitination and degradation of the fusion protein,
IKZF1/mNeon (or the fusion protein, IKZF3/mNeon). Regarding
doxycycline, doxycycline was used at 3 micromolar in order to
induce expression of the lentiviral vector construct. A
concentration/induction curve with doxycycline is shown by Go and
Ho (2002) J. Gene Medicine. 4:258-270). After transfection with the
lentivirus vector, the following condition was used to keep IKZF1
minimally expressed in growing cells. The condition was to leave
doxycycline out of the medium, and also to use "insulating
sequences" in the construct. The insulating sequences prevent
read-through from promoters outside of the construct. Insulating
sequences have been described (see, Anton et al (2005) Cancer Gene
Therapy. 12:640-646; Carr et al (2017) PLoS ONE. 12:e0176013).
Insulating sequences prevent promoters that are outside of the
construct from driving expression of an open reading frame (ORF)
that is part of the construct. To put cells into picowells, cells
can be transferred to the top surface of a picowell plate, at a
given ratio of, [number of cells]/[number of picowells]. The ratio
can be, for example, about 1 cell/40 wells, about 1 cell/20 wells,
about 1 cell/10 wells, about 2 cells/10 wells, about 4 cells/10
wells, about 8 cells/10 wells, about 16 cells/10 wells, about 32
cells/10 wells, about 50 cells/10 wells, about 100 cells/10 wells,
and so on. The cells can be used for assays in picowells as soon as
cells attach to the vitronectin that coats the bottom of the
picowell.
[0551] Details of lentivirus construct and cell culture. This
concerns onstructing reporter cell lines for IKZF1/3, culturing
them in picowells, and assaying them with bulk lenalidomide. The
plasmids carrying reporter construct were assembled from parts
using Gibson assembly (see maps attached). Lentivirus with reporter
construct, as well as UbC driven rtTA-M2.2 were made in LentiX
HEK293T cells (Clontech, Palo Alto, Calif.) with 3.sup.rd
generation packaging system (chimeric CMV promoter and no tat
protein). The plasmids were transfected via calcium precipitation
method. Virus supernatant was harvested in the recommended LentiX
media plus 1% bovine serum albumin (BSA), and filtered through 0.45
um low protein bind filters (Millipore). The host HeLa cells were
obtained from ATCC, cultured in standard conditions. Viral
supernatant was applied to sub-confluent HeLa culture, after 24
hours changed to LentiX media with Doxicyclin. Two days before
clone selection, lenalidomide was added to the culture. Clones were
selected via fluorescence activated cell sorting (FACS), gated on
both AlexaFluor 488 (negative) and Cy3 channels (positive). Clones
were grown for 10 days without lenalidomide before assays. The most
stable expression level clones are used for screening.
[0552] This describes experiment to seal cells with beads and lyse
cells through porous beads. 96 well plate with picowell patterned
bottom (MuWells) is treated with Pluronic F127 detergent
(Sigma-Aldrich, St. Louis, Mo.) without vacuum applied to passivate
upper part of the wells. After 30 min incubation, excess of
detergent is washed away with phosphate buffered saline (PBS) or
distilled H.sub.2O. Wells are flushed with ethanol and dried in the
biosafety cabinet with the air flow. Wells are wetted with PBS
under strong vacuum to a completion, and PBS is replaced with
Virtonectin coating reagent (Preprotech). The plate is incubated
for 30 min at 37 C. Vitronectin coating reagent is removed and
reporter cells are seeded at desirable density. From the moment of
cell seeding, media stays in the dish throughout the assay.
TentaGel.RTM. beads carrying the photocleavable compound could be
seeded before vitronectin coating, or after cell seeding. PEG
polymer beads are loaded on top of the culture in the excess over
the well number. Spin the plate at 400rcf for 1 min. Photo-release
the compound off the beads using 365 nm LED light source for
appropriate amount of time. Incubate in the CO2 incubator until the
imaging (readout of the fluorescent reporters).
[0553] Constructs. FIG. 20 and FIG. 21 disclose the relevant
constructs. Each of these figures discloses the sequence that is to
be integrated into the HeLa cell genome, and each of the figures
discloses the carrier sequence (the sequence belonging to
lentivirus). Sequence belonging to lentivirus is from about one
o'clock to about nine o'clock, where this sequenced is bracketed by
two long terminal repeats (LTRs). Sequence from about nine o'clock
to about one o'clock gets integrated into HeLa cell genome. In
detail, first a plasmid is transfected into producer cells (HEK93T)
(Clontech, Palo Alto, Calif.). The producer cells produce and then
release lentivirus. The released lentivirus then infects HeLa cells
and integrates nucleic acids into the HeLa cell genome.
[0554] Optics. For the present cell culture experiments, Applicants
used EBQ100 Isolated mercury lamp connected to HBO 100 (Carl Zeiss
Microscopy, GmbH, Germany), which was connected to an Axiovert
200-M Carl Zeiss microscope with Ludl Electronic Products stage
(Ludl Electronic Products, Ltd., Hawthorne, N.Y.). Applicants also
used filter cubes with mercury lamp, where filter cubes controlled
wavelength of excitation and also controlled wavelength of
detecting emission. Images were captured with Basler ACA2440-35UM
(Basler AG, 22926, Ahrensburg, Germany). Halogen lamp was used, as
an alternative to mercury lamp. Microwell plates, picowell plates,
and the like, were held in place with a plate holder and an "XY
stage" with controller. XY stages and other precise positioning
stages for optics use are available from, Newmark Systems, Inc.,
Rancho Santa Margarita, Calif.; Aerotech, Inc., Pittsburgh, Pa.,
Physik Instrumente GmBH, 76228 Karlsruhe, Germany.
Example 5. Mdm2-Based Assay for Active Compounds
[0555] Modifying glass to contain an amino group. Silica substrates
can be modified to contain an amino group, by way of one or more of
a number of "functional silanes." These "functional silanes" are
3-aminopropyl-triethoxysilane (APTES),
3-aminopropyl-trimethoxysilane (APTMS),
N-(2-aminoethyl)-3-aminopropyltriethoxysilane (AEAPTES),
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPTMS), and
N-(6-aminohexyl)aminomethyltriethoxysilane (AHAMTES). Reactions of
these reagents with glass can be conducted in a vapor phase or in a
solution phase (see, Zhu, Lerum, Chen (2012) Langmuir.
28:416-423).
[0556] Results from biochemical assays of compounds (MDM2-based
assay). Laboratory methods. The following reagent was applied to a
glass slide. The glass slide was modified to have amino groups. The
reagent was NHS-PEG-mTET. NHS is N-hydroxy-succinimide. NHS is a
type of activated ester. NHS is useful in bioconjugation reactions,
such as surface activation of microbeads or of microarray slides
(Klykov and Weller (2015) Analytical Methods. 7:6443-6448).
[0557] PEG is polyethylene glycol. mTET is methyltetrazine. This
reagent was mixed with DMSO, and then a volume of 2 microliters was
applied to the glass slide. The mixture was made by mixing 10
microliters of 50 mM NHS-PEG-mTET with 30 microliters DMSO. The NHS
group reacts with the amino groups of the glass side, where the
result is that the mTET group is affixed to the glass slide. The
goal of the mTET was to create a covalent link between the slide
and the bead.
[0558] TCO and tetrazine can mediate "click chemistry" reactions.
Examples of these click chemistry reactions, is using antibodies
that are functionalized with tetrazine to couple with DNA that is
functionalized by TCO. Or using antibodies modified with TCO to
couple with tetrazine-modified beads (see, van Buggenum et al
(2016) Scientific Reports. 6:22675 (DOI:10.1038); Rahim et al
(2015) Bioconjug. Chem. 18:352-360; Haun et al (2010) Nature
Nanotechnol. 5:660-665).
[0559] In detail, the glass slide was prepared by applying a sheet
of parafilm to the top of the slide, where the parafilm had an
aperture cut out of the middle, where the drop of the above mixture
was applied in the aperture directly to the glass slide. Before
applying the mixture, the glass slide with the parafilm on top was
heated at full heat for 90 seconds, in order to create a tight seal
between the parafilm and the slide, in order to prevent seepage of
liquids after applying the mixture to the open area (the aperture)
in the parafilm. The glass slide, with the 2 microliter droplet
sitting in the aperture cut into the Parafilm, was incubated
overnight at room temperature. During the incubation, the glass
slide was inside a petri dish, where the dish was covered with a
glass cover that covered the top and sides of the petri dish.
Before the overnight incubation, a square of Parafilm was placed
over the drop and over the surrounding Parafilm, in order to
prevent water from evaporating from the drop.
[0560] Inventive method to make complex of slide/bead/antibody.
Applicants' method used beads that were functionalized by TCO. The
TCO groups of the bead mediated covalent attachment of the
methyltetrazine-functionalized slide to the bead. Also, the TCO
groups of the bead mediated covalent attachment of the
methyltetrazine-functionalized anti-p53 antibody to the bead.
[0561] Applicants surprisingly found that, if the first step is to
contact slide and bead, then subsequent addition of antibody will
NOT result in covalent attachment of the antibody to the bead.
Also, Applicants surprisingly found that, if the first step is to
contact bead with antibody, then subsequent transfer of this
mixture to the slide will NOT result in covalent attachment of the
bead to the slide. In a preferred method, all of these three
reagents--the slide, the bead, and the antibody--are simultaneously
brought into contact with each other. In another preferred
embodiment, the bead and antibody are first mixed together to
initiate covalent linking of the bead to the antibody, and then
immediately or within a few minutes, this mixture is applied to the
slide, where the result is covalent linking of the bead to the
slide.
[0562] Nature of the enzyme-based screening assay. The assay takes
the form of a glass slide with an attached bead. The bead contains
attached antibodies that are specific for binding to the
transcription factor, p53. This antibody can bind to human p53 and
also to ubiquitinated human p53. So far, it can be seen that the
assay method involves a sandwich between the following
reagents:
[0563] Slide/Covalently bound bead/Bead-bound anti-p53
Ab/Ubiquitinated p53
[0564] The readout from this assay is ubiquitinated-p53, where the
ubiquitinated-p53 is detected by a fluorescent antibody that is
specific for ubiquitin. In detail, the antibody is a polyclonal
antibody made in the goat, where the antibody is tagged with a
fluorophore (AF488). FIG. 8 discloses the structure of AF488. This
fluorescent antibody binds to ubiquitin. Thus, when
ubiquitinated-p53 is detected, what exists is the following
sandwich:
[0565] Slide/Covalently bound bead/Bead-bound bound anti-p53
Ab/Ubiquitinated p53/Fluorescent Ab
Example 6. Sequencing DNA in Picowells
[0566] Sequencing of bead-bound DNA barcodes was performed, where
beads were situated in a picowell, one bead per picowell. The assay
method involved interrogating each position on the bead-bound DNA
barcode, one at a time, by way of transient binding of fluorescent
nucleotides. Each bead contained about one hundred attomoles of
coupled DNA barcode, where coupling was by click-chemistry. This
number is equivalent to about sixty million oligonucleotides,
coupled per bead. For each base on the DNA barcode, the assay
involves adding all four fluorescent dNTPs at the same time.
Without implying any limitation, the four fluorescent dNTPs were
AF488-dGTP, CY3-dATP, TexasRed-dUTP, and CY5-dCTP. Fluorescent
signals were captured, and then processed by ImageJ software
(National Institutes of Health, NIH), to provide a corresponding
numerical value. The data are from sequencing five consecutive
nucleotides (all in a row) that was part of the bead-bound DNA
barcode. The bead-bound DNA barcode included a DNA hairpin region.
The bases in the DNA hairpin region annealed to itself, resulting
in the formation of the hairpin, and where the 3'-terminal
nucleotides in this DNA hairpin served as a sequencing primer.
Sequencing by transient binding was initiated at this 3'-terminus.
The sequencing assay was performed in triplicate, that is, using
three different beads, where one DNA barcode sequence was used for
each of the three beads. In other words, each of the three beads
was expected to provide a sequencing read-out identical to that
provided by the other two beads.
[0567] FIG. 28 discloses sequencing results, where sequencing was
conducted on bead-bound DNA barcode. What is shown are results from
interrogating the first base, the second base, the third base, the
fourth base, and the fifth base. For each of these bases, what is
separately shown, by way of separate histogram bars, is the
fluorescent emission produced with interrogation with AF488-dGTP,
CY3-dATP, TexasRed-dUTP, and CY5-dCTP, respectively. Each of the
four histogram bars has different graphics: AF488-dGTP (black
outline, gray interior), CY3-dATP (black outline, white interior),
TexasRed-dUTP (solid black histogram bar), and CY5-dCTP (solid gray
histogram bar). The bead diameter was 10-14 micrometers, after
swelling in aqueous solution. The volume of the picowell was 12
picoliters.
[0568] The template sequence that was interrogated was:
5'-CTCACATCCCATTTTCGCTTTAGT-3' (SEQ ID NO: 1). For this particular
sequencing assay, five consecutive bases were interrogated, where
the fluorescent dNTPs that gave the biggest fluorescent signal were
fluorescent dGTP, dATP, dGTP, dUTP, and dGTP, which corresponds to
a sequence on the template that is dC, dT, dC, dA, and dC. Thus,
the sequencing results were 100% accurate. The results demonstrate
that the bead-bound DNA barcodes can be sequenced, that is, when
the DNA barcode is still bound to the bead. In other words, the
bead-bound DNA barcodes are sequencable.
Example 7. Cell Barcoding
[0569] Introduction to the concept of barcoding. This introduces
the concept of barcoding. A common barcoding technique is barcoding
the transcriptome of a given single cell. FIG. 36 and FIG. 37
illustrate steps for procedures where the transcriptome is captured
and amplified, in preparation for future sequencing. FIG. 36 shows
lysis of cells to release mRNAs, followed by reverse transcription.
FIG. 37 shows capture of mRNAs by way of immobilized poly(dT),
followed by reverse transcription, and finally sequencing.
Sequencing can be with Next Generation Sequencing (NGS).
[0570] Some or most of the messenger RNA (mRNA) molecules from a
given cell can be tagged with a common barcode, where this tagging
allows the researchers to determine, for any given mRNA sequence,
the origin of that coding sequence in terms of a given cell. For
example, where nucleic acids representing each of the separate
transcriptomes from one hundred different single cells are mixed
together, and where the nucleic acids from each of the 100
different single cell has its own barcode, then the following
advantage will result. The advantage is that nucleic acids from all
of the transcriptomes can be mixed together in one test tube, and
then subjected to Next Generation Sequencing, where the barcode
enables the user to identify which information is from the same
cell.
[0571] The above advantage is described in a different way, as
follows. In using mRNA barcoding, a given single cell is processed
so that information from some or most of the mRNA molecules from
that cell are converted to corresponding molecules of cDNA, where
each of these cDNA molecules possesses exactly the same DNA
barcode. This barcoding procedure can be repeated with ten, twenty,
100, several hundred, or over 1,000 different cells, where the cDNA
molecules from each of these cells is distinguished by having a
unique, cell-specific barcode. This method enables the researcher
to conduct DNA sequencing, all in one sequencing run, from a pool
of all of the barcoded cDNA molecules from all of the cells (all
barcoded cDNA molecules mixed together, prior to sequencing) (see,
Avital, Hashimshony, Yanai (2014) Genome Biology. 15:110).
[0572] Barcodes that tag nucleic acids compared with barcodes that
tag the plasma membrane. Guidance is available for preparing
libraries of chemicals, where each chemical, or where all members
of each class of chemicals, is associated with a unique DNA barcode
(see, Brenner and Lerner (1992) Proc. Nat'l. Acad. Sci.
89:5381-5383; Bose, Wan, Carr (2015) Genome Biology. 16:120. DOI
10.1186). With the above barcoding example in mind, the following
provides another type of barcoding which can also be applied to a
particular, single cell. The present disclosure provides
cell-associated barcoding that takes the form of a tag that is
stably attached to the cell's plasma membrane.
[0573] Option of at least two kinds of barcodes that get attached
to the plasma membrane-bound. A barcode used for tagging the plasma
membrane of given cell can include a first barcode that identifies
the type of cell, and a second barcode that identifies a perturbant
that was exposed to the cell. For example, the first barcode can
identify the cell as originating from a healthy human subject,
Human Subject No. 38 from Clinical Study No. 7, a human primary
colorectal cancer cell line, a five-times passaged human primary
colorectal cancer cell line, a multiple myeloma human subject with
multiple myeloma, a treatment-naive Human Subject No. 23 with
multiple myeloma, or from a treatment-experienced Human Subject No.
32 with multiple myeloma.
[0574] Also, the barcode can identify a "perturbant" that was given
to that particular single cell (given either before or after
barcoding). The "perturbant" can be an anti-cancer drug, a
combination of anti-cancer drugs, a combinatorially generated
compound, or a combination of an antibody drug and a small molecule
drug. The barcoding can be used to keep track of a given single
cell, and can be used to correlate that cell with subsequent
behaviors such as activation or inhibition with one or more
cell-signaling pathways, increased or decreased migration,
apoptosis, necrosis, change in expression of one or more CD
proteins (CD; cluster of differentiation), change in expression of
one or more oncogenes, change in expression of one or more
microRNAs (miRNAs). Expression can be in terms of, transcription
rate, level of a given polypeptide in the cell, change in location
of a given protein from cytosolic to membrane-bound, and so on.
[0575] Tagging cell-surface oligosaccharides of membrane-bound
glycoproteins. Methods and reagents are available for connecting
tags, such as DNA barcodes, to the plasma membrane of a living
cell. Tagging can be accomplished with a reagent consisting of a
covalent complex of a DNA barcode with a reactive moiety that
attacks and covalently binds to oligosaccharide chains of
membrane-bound glycoproteins. The literature establishes that
hydrazide biocytin can be used to connect biotin to carbohydrates
on membrane-bound glycoproteins. The present disclosure uses this
reagent, except with the biotin replaced with a DNA barcode. The
carbohydrate needs to be oxidized to form aldehydes. The hydrazide
reacts with the aldehyde to form a hydrazine link. The sialic acid
component on the oligosaccharides is easily oxidized with 1 mM Na
meta-periodate (NaIO4). In conducting the oxidation step, and
hydrazide-linking step, buffers with a primary amine group should
be avoided. See, for example, "Instructions. EZ-LinkHydrazide
Biocytin. Number 28020. ThermoScientific (2016) (4 pages), Bayer
(1988) Analyt. Biochem. 170:271-281; Reisfeld (1987) Biochem.
Biophys. Res. Commun. 142:519-526, Wollscheid, Bibel, Watts (2009)
Nature Biotechnol. 27:378-386.
[0576] Another method for tagging the oligosaccharide moiety of
glycoproteins on living cells, is to use periodate oxidation and
aniline-catalyzed oxime ligation. This method uses mild periodate
oxidation of sialic acids and then ligation with an aminoxy tag in
the presence of aniline. In a variation of this method, galactose
oxidase can be used to introduce aldehydes into terminal galactose
residues and terminal N-acetylgalactosamine (GalNAc) residues of
oligosaccharides. Galactose oxidase catalyzes the oxidation, at
carbon-6, to generate an aldehyde. Following aldehyde generation,
one can couple with aminoxybiotin using aniline-catalyzed ligation
(see, Ramya, Cravatt, Paulson (2013) Glycobiology. 23:211-221). The
present disclosure replaces the biotin with a DNA barcode and
provides aniline-catalyzed ligation of an aminoxy-DNA barcode.
[0577] Tagging mediated by an antibody bound to the cell surface.
The present disclosure provides methods and reagents for attaching
barcodes to the plasma membrane of a cell, where attachment is
mediated by an antibody that specifically binds to a membrane-bound
protein. The antibody can be covalently modified with
trans-cyclooctene (TCO) where this modification can be conducted
with an overnight incubation at 4 degrees C. (see, Supporting
Information (5 pages) for Devaraj, Haun, Weissleder (2009) Angew.
Chem. Intl. 48:7013-7016). This covalent modification of antibody
can be carried out with the reagent, trans-cyclooctene succinimidyl
carbonate (Devaraj, Haun, Weissleder (2009) Angew. Chem. Intl.
48:7013-7016). The antibody-tetrazine complex can then be contacted
with a cell, resulting in membrane-bound antibodies. The
membrane-bound antibodies each bear a tetrazine moiety, which
enables tagging of the antibody via click chemistry, such as, by
exposing the antibodies to a DNA barcode-tetrazine complex.
[0578] Tetrazine can be introduced at free amino groups of the
antibody, using the reagent, N-hydroxysuccinimide ester (NETS)
(see, van Buggenum, Gerlach, Mulder (2016) Scientific Reports.
6:22675). Once the antibody contains one or more tetrazine groups,
the antibody can be further modified by attaching a DNA barcode, by
way of a reagent that is TCO-DNA barcode. With this modified
antibody in hand, the antibody can then be used for a tagging
living cell, where the antibody binds to a membrane-bound protein
of the cell.
[0579] A complex of tetrazine-DNA barcode can be prepared. This
complex can then be introduced into a cell medium, where the medium
includes cells, and where the cells bear the attached antibody-TCO
complex. Where the tetrazine-DNA barcode contacts the
membrane-bound antibody-TCO complex, the result is a click
chemistry reaction where the cells become tagged with the DNA
barcode. This click chemistry reaction can be carried out for 30
minutes at 37 degrees C.
[0580] Preferred antibodies for use in the above procedure are
those that bind tightly and specifically to membrane-bound proteins
of the plasma membrane, where the membrane-bound protein occurs in
high abundance, for example, at over 50,000 copies per cell
membrane, and where the membrane-bound protein is stable on the
cell surface and does not much recycle into the cell's interior,
and where the membrane-bound membrane does not much shed into the
culture medium.
[0581] Tagging membrane-bound proteins with azide followed by click
chemistry with an octyne conjugate. Azide can be introduced on
membrane-bound proteins of a living cell by way of the enzyme,
lipoic acid ligase, followed by attachment of a fluorinated octyne
compound that is conjugated to a DNA barcode. The conjugation of a
fluorinated octyne compound to a fluorophore is described (see,
Jewett and Bertozzi (2010) Chem. Soc. Rev. 39:1272-1279;
Fernandez-Suarez, Bertozzi, Ting (2007) Nature Biotechnol.
25:1483-1487). To reiterate, "Ting and co-workers introduced azides
into mammalian cell-surface proteins using . . . lipoic acid ligase
. . . [t]he protein could then be labeled with a fluorinated
cyclooctyne-conjugated fluorescent dye-conjugated fluorescent dye"
(Jewett et al, supra).
Example 7. Caps Over Picowells
[0582] Capping picowells. Each picowell was capped with a sphere,
one sphere to each picowell, where the sphere fits into the
aperture (top opening) of the picowell. To apply the spheres to the
picowell plate, the spheres are put into growth media and
suspended, then applied to the top surface of the picowell plate,
and the sphere allowed to settle. Then, the entire plate is placed
in a centrifuge and spun at a low-gravity, in order to get a firm
sitting of the spheres in the aperture of each picowell.
[0583] Active caps and passive caps. FIG. 18A shows an active cap
inserted into the top of a picowell, and FIG. 18B shows a passive
cap inserted into the top of a picowell. Preferably, the caps are
made of material that is softer than the material used to make the
picowell plate, where the result is slight deformation of the cap
when it is pressed into the aperture of the picowell, and where the
result is a snug fit that prevents leakage. In embodiments, the
present disclosure provides one or more of active caps, passive
caps, or both active caps and passive caps. Each cap may be
free-standing and not connected to any other cap. In an alternative
embodiment, to more caps may be connected together, for example, by
way of a sheet of polymer that is capable of being layed upon the
top surface of the plate, and where a plurality of caps protrude
from the bottom of the sheet of polymer, and where the protruding
caps are predeterminedly spaced in order to fit into each picowell.
An active cap may be used instead of a bead that is capable of
sitting on the floor of a microwell. The active cap contains many
attached copies of substantially identical compounds, where each
compound is attached to the active cap (shown here in the sample of
a spherical bead), and where cleavage results in release of the
compounds into the solution that resides in the microwell (FIG.
18A).
[0584] Regarding the passive cap, the passive cap is porous and it
acts like a sponge. It absorbs products from biochemical reactions,
and thus facilitates collection of products where the goal of the
user is to determine the influence of a given compound on living,
biological cells that are cultures in the picowell. In other words,
the compound stimulates the cells to respond, where the response
takes the form of increased (or decreased) expression of one or
more metabolites, and where some of the metabolites diffuse towards
the passive cap and are absorbed by the passive cap. The user can
then collect the passive caps and analyze the metabolites that had
absorbed to the passive cap (FIG. 18B).
[0585] Polymer mat that adheres to an array of caps. FIGS. 19A-19D
illustrate a polymer mat that is capable of adhering to each cap in
an array of porous caps. Once adhered, the polymer mat can be
peeled away and removed, bringing with it each porous cap in the
array. As a result, the polymer mat with the porous caps can be
used for assays that measure metabolites or other chemicals that
are associated with the porous cap.
[0586] To provide a step-wise example, each well in an array of
many thousands of picowells can contain one bead, where each bead
contains one type of compound, where the compound is attached via a
cleavable linker. The picowell also contains a solution as well as
cultured cells. The picowell is sealed with a porous cap, and where
the porous cap contacts the solution and is able to capture
(sample; absorb; absorb) metabolites that are released from the
cultured cells. The metabolites can be metabolites of the compound,
or the metabolites can take the form of cytokines, interleukins,
products of intermediary metabolism, microRNA molecules, exosomes,
and so on. Finally, a solution of polyacrylamide is poured over the
picowell plate, and the polyacrylamide allowed to soak into the
thousands of porous caps, and then solidify in the form of a mat
that is firmly adhered to each and every one of the caps. The
solified mat is then removed, where each cap is separately analyzed
for absorbed metabolites.
[0587] In preferred embodiments, a polyacrylamide gel is used to
crosslink the capping beads into the enmeshing layer or the mat.
The protocol to create an 20% solution of polyacrylamide solution
that can be poured over the picowell array to cure and enmesh the
capping bead is as follows. Add 4 ml of a 40% bis-acrylamide
solution and 2 ml of 1.5 M Tris pH 8.8 to 1.8 ml distilled
deionized water. Just before pouring this mixture over the capped
picowell array, 80 microliters of the free radical initializer
ammonium persulfate (APS, 10% stock solution), and 8 microliter of
the free radical stabilizer N,N,N',N'-tetramethylethylene-diamine
(TEMED) are added to begin crosslinking of the gel. The gel layer
is poured before complete crosslinking and allowed to fully
crosslink over the capped picowell array. One fully crosslinked
(stiff enough to be handled, or roughly 60 minutes of setting), the
polyacrylamide layer may be peeled off using tweezers. It is found
that the capping beads are lifted off the tops of the picowells and
get attached to the polyacrylamide layer. This behavior can be
observed for multiple bead types including polyacrylamide beads,
Tentagel beads, polystyrene beads and silica beads.
[0588] Measuring efficacy of cap in preventing leaks. In
embodiments, the efficacy of a cap can be determined by using the
bead with the photocleavable linker. Images of a picowell, or of
several picowells in one particular picowell array can be captured
just before exposing picowells to UV light, and in the time frame
after exposing picowells to UV light. For example, images can be
captured at t=minus ten seconds and at t=10 seconds, 20 sec, 40
sec, 60 sec, 2 minutes, 4 min, 8 min, 15 min, 60 min, 90 min, 2
hours, 3 hours, and 4 hours. Excellent efficacy can be shown where
the fluorescence of a given well at 2 hours is equal to at least
90%, at least 95%, at least 98%, or about 100% the fluorescence
found at t=10 seconds, with subtraction of the background image
taken at t=minus ten seconds. Images can also be taken of a region
of the picowell plate outside of the picowell, for example, in the
immediate vicinity of the cap. Excellent efficacy can be shown
where the fluorescence of an area on the surface of the plate
(outside of the picowell) and in the immediate vicinity of the cap
is less than 1%, less than 0.5%, less than 0.1%, less than 0.05%,
less than 0.01%, less than 0.005%, or less than 0.001%. This
comparison may be made without regard to the volume of the fluid in
the well, and without regard to the volume of any fluid situated on
top of the plate and outside of the cap, and here, the comparison
may simply take into account the entire visual field that is
captured by the light detector. Alternatively, the comparison may
be made with correction of the depth of the fluid (depth of
picowell; depth of fluid on top of the picowell plate). Also
alternatively, the comparison may take into account diffusion of
any leaking fluorophore over the entire surface of the picowell
plate.
[0589] How barcoding fits into the reagents and methods of the
present disclosure. The following provides further embodiments of
the reagents and methods of the present disclosure.
[0590] Reagents and capabilitites. A microscopic bead is provided.
The microscopic bead can be covalently modified by a plurality of
first linkers, each capable of coupling by way of solid-phase
synthesis with monomers, where completion of the solid-phase
synthesis creates a member of a chemical library. This member of
the chemical library is bead-bound. The same microscopic bead can
be covalently modified by a plurality of second linkers, each
capable of being coupled with a plurality of DNA barcodes. This
member of the DNA barcode is bead-bound.
Example 9. DNA Barcode of the Present Disclosure
[0591] This concerns a set of information that can be printed on
paper, or stored in computer language, that provides a "DNA
barcode" that correlates a DNA sequence with a chemical library
member. This DNA barcode may be called a "legend" or a "key." The
DNA barcode also provides nucleic acids that can identify a
specific class of chemical compounds, such as analogs of a specific
FDA-approved anti-cancer drugs, or that can identify the user's
name, or that can identify a specific disease that is to be tested
with the bead-bound chemical library.
Example 10. Lenalidomide Analogs
[0592] FIGS. 13, 14, and 15 disclose the conversion of lenalidomide
to three different derivatives, each derivative bearing a
carboxylic acid group. Each of these carboxylic acid groups can
subsequently be used to condensed with the bead-linker complex. In
this situation, where the carboxylic acid group is condensed to the
bead-linker complex, it is attached at the position that was
previously occupied by Fmoc.
[0593] Starting with a primary amine and converting it to a
carboxylic acid (FIG. 13). Applicants take the approach of
generating a library of compounds by converting a compound with a
primary amine to a compound with a carboxyl group. FIG. 13
discloses starting with lenalidomide. Lenalidomide has a primary
amine. To this is added, succinic anhydride in
4-dimethylaminopyridine (DMA) and acetonitrile (ACN). The succinic
anhydride condenses with the primary amino group, resulting in
lenalidomide bearing a carboxylic acid group. The term "cat." in
the figure means, catalytic.
[0594] Subsequently, this carboxylic acid group can be linked to a
bead. Thus, the resulting complex is: BEAD-succinic acid
moiety-lenalidomide
[0595] FIG. 14 discloses starting with linalidomide and adding
t-butyl-bromoacetate, to give an intermediate. The intermediate is
then treated with FmocOSu (o-succinimide), to produce a final
product that is a carboxylic acid derivative of lenalidomide. The
carboxylic acid moiety can then be condensed with a free amino
group, for example, with the free amino group that once had an
attached Fmoc group. Alternatively, the carboxylic acid can be
condensed with the free amino group of a chemical monomer residing
on the bead, where the result of the condensation is two chemical
monomers attached to each other.
[0596] FIG. 15 discloses lenalidomide as the starting material. The
lenalidomide is reacted with 3-carboxybenzaldehyde, where the
aldehyde group condenses with the amino group, resulting in yet
another type of carboxylic acid derivative of lenalidoimide.
[0597] FIG. 16A, FIG. 16B, and FIG. 16C discloses yet another
approach of Applicants for generating a library of novel and unique
bead-bound compounds, where compounds can be released from the
bead, and then tested for activity in cell-based assays or in
cell-free assays. Each of the three compounds is a lenalidomide
analogue, where the primary amine is in a unique position of the
benzene ring.
Example 11. Picowells Containing Cells Together with Beads that
have a Coupled Response-Capture Element
[0598] The present disclosure provides reagents, systems, and
methods for assessing response of a cell to a compound, and where
response that is measured takes the form of changes in the
transcriptome. "Changes in the transcriptome" can refer, wtihout
implying any limitation, to change in amount each and every type of
unique mRNA in the cell, and well as to change in amount of a
pre-determined set of mRNA molecules in the cell. "Changes in
transcriptome" includes change from below the lower limit of
detection to becoming detectable, as well as change from being
detectable to dropping below the lower limit of detection, where
these changes are associated with release of the bead-bound
compound.
[0599] Cells can be lysed by adding detergent or surfactant to the
picowell array. For example, a volume of buffer containing
detergent can be pipetted into a microwell that contains, within
it, many thousands of picowells. The detergent can be allowed to
diffuse into all of the picowells, causing lysis of the cells
within, release of mRNA, and finally binding by the bead-bound
"capture response element."
[0600] Cell lysis. Cells can be lysed by one or more cycles of
freezing and thawing (Bose, Wan, Carr (2015) Genome Biology.
16:120. DOI 10.1186). Cells can also be lysed with
perfluoro-1-octanol with shaking (Macosko, Basu, Satija (2015)
Cell. 161:1202-1214; Ziegenhain (2017) Molecular Cell. 65:631-643;
Eastburn, Sciambi, Abate (2014) Nucleic Acids Res. 42:e128). Also,
cells can be lysed by a combination of a surfactant (Tween-20.RTM.)
and a protease (Eastburn, Sciambi, Abate (2013) Anal. Chem.
85:8016-8021). Lysis of cells results in release of mRNA. The mRNA
is captured by the bead that resides in the same picowell as the
lysed cell (or cells). The bead contains a huge number of
bead-bound polynucleotides, where each polynucleotide contains two
nucleic acid, where the first nucleic acid contains a common DNA
barcode and the second nucleic acid contains a "response capture
element." Where the goal is indiscriminate capture of all mRNAs in
the cell, the "response capture element" can take the form of
poly(dT). This poly(dT) binds to the poly(A) tail of the mRNA
molecules.
[0601] More cell lysis conditions. Cell lysis can be effected by
exposure to detergent with a sodium salt, for example, 0.05% Triton
X-100 with 15 mM NaCl, 25 mM NaCl, 50 mM NaCl, 75 mM NaCl, 100 mM
NaCl, 0.1% Triton X-100 with 15 mM NaCl, 25 mM NaCl, 50 mM NaCl, 75
mM NaCl, 100 mM NaCl, 0.2% Triton X-100 with 15 mM NaCl, 25 mM
NaCl, 50 mM NaCl, 75 mM NaCl, 100 mM NaCl, or 0.5% Triton X-100
with 15 mM NaCl, 25 mM NaCl, 50 mM NaCl, 75 mM NaCl, 100 mM NaCl,
or with detergent with a potassium salt, such as, 0.05% Triton
X-100 with 15 mM KCl, 25 mM KCl, 50 mM KCl, 75 mM KCl, 100 mM KCl,
0.1% Triton X-100 with 15 mM KCl, 25 mM KCl, 50 mM KCl, 75 mM KCl,
100 mM KCl, 0.2% Triton X-100 with 15 mM KCl, 25 mM KCl, 50 mM KCl,
75 mM KCl, 100 mM KCl, or 0.5% Triton X-100 with 15 mM KCl, 25 mM
KCl, 50 mM KCl, 75 mM KCl, 100 mM KCl. Exposure can be for 10 min,
20 min, 40 min, or 60 min at about 4 degrees C., or at room
temperature (23 degrees C.), and so on.
[0602] Present disclosure can assess influence of a compound on an
expression profiles. A bead-bound capture element can take the form
of one or more deoxyribonucleotides that can specifically hybridize
to one or more mRNA molecules of interest, where the one or more
mRNA molecules are associated with a specific disease. Expression
profiles for various diseases are available, for example, for colon
cancer (Llarena (2009) J. Clin. Oncol. 25:155 (e22182), ovarian
cancer (Spentzos (2005) J. Clin. Oncol. 23:7911-7918), and lung
adenocarcinoma (Takeuchi (2006) J. Clin. Oncol. 11:1679-1688). To
give a similar example, what can also be characterized is the
influence of a released compound on mRNAs associated with
non-hepatic tumor cells that have metastasized to the liver (see,
Barshack, Rosenwald, Bronfeld (2008) J. Clin. Oncol. 26:15 Suppl.
11026, Barshack (2010) Int. J. Biochem. Cell Biol.
42:1355-1362.).
[0603] Capturing the transcriptome. Methods are available for
capturing mRNA by hybridizing their polyA group to immobilized
poly(dT) (see, Dubiley (1997) Nucleic Acids Res. 25:2259-2265;
Hamaguchi, Aso, Shimada (1998) Clinical Chem. 44:2256-2263; D.S.
Hage (2005) Handbook of Affinity Chromatography, 2nd ed, CRC Press,
page 549).
[0604] After capture of mRNA molecules released from the lysed cell
(or cells), the bead-bound polynucleotide serves as a primer that
supports reverse transcription from the mRNA, resulting in a
bead-bound complementary DNA (cDNA), and where this bead-bound cDNA
can be sequenced. Alternatively, the bead-bound cDNA can be
released from the bead, where the bead-bound "response caputure
element" is coupled to the bead with a cleavable linker, such as
with a photocleavable linker. If a photocleavable linker is used,
cleaving conditions for releasing bead-bound compounds (compounds
made from a chemical monomer library) but not also cleave the
bead-bound "response capture element."
[0605] Where cells are exposed to a bead-bound compound or to a
compound released from a bead, cells can be screened for a genetic
response, for example, by characterizing any changes in the
transcriptome with or without exposure to the compound. Also, cells
can be screened for a phenotypic response, for example, apoptosis,
change in activity of one or more cell-signaling proteins, or
change in cell-surface expression of one or more CD proteins. CD is
Cluster of Differentiation (See, Lal (2009) Mol. Cell Proteomics.
8:799-804; Belov (2001) Cancer Res. 61:4483-4489; IUIS/WHO
Subcommittee on CD Nomenclature (1994) Bull.World Health Org.
72:807-808; IUIS-WHO Nobenclature Subcommittee (1984) Bull.World
Health Org. 62:809-811). For some phenotypic response assays, the
cells must not be lysed.
[0606] The present disclosure addresses the unmet need to partition
different drugs to different cells, for example, by exposing a
single cell to one type of drug where exposure occurs in a
picowell.
[0607] The present disclosure also eliminates the need to prepare
barcoded mRNA, where mRNA is released from a cell followed by
preparing cDNA (in this type of barcode, all mRNA from a given cell
receives the same barcode, when the transcriptosome is coverted to
corresponding library of cDNA).
[0608] Parameters during cell incubation with the perturbant. For
any given compound or some other type of perturbant, parameters
that can be varied or controlled light, temperature, pH of cell
medium, sound, concentration and exposure time to a reagent
(reagent can be the compound released from the bead, an enzyme
substrate, a cytokine, a compound that is already an established
drug, a salt), mechanic agitation, an antibody against a
cell-surface protein, and so on.
[0609] Barcoding the cell. Cells can be incubated with a bead-bound
compound or with the compound following cleavage from a bead-bound
cleavable linker. During or after incubation, cells can be barcoded
with a membrane-bound barcode that identifies the purturbant. This
membrane-bound barcode can be coupled to oligosaccharides of the
cell membrane, polypeptides of the cell membrane, or phospholipids
of the cell membrane.
[0610] Response capture elements other than poly(dT). Messenger RNA
can be captured by way of the 5-prime 7-methylguanosine cap. This
method is especially useful where there polyA tail is short (see,
Blower, Jambhekar (2013) PLOS One. 8:e77700). Also, mRNA can be
captured using immobilized DNA that is specific for a coding region
of the mRNA. This method is called, "RNA exome capture," and
variations of this name. According to Cieslik et al, "Unique to
capture transcriptomics is an overnight capture reaction (RNA-DNA
hybridization) using exon-targeting RNA probes" (Cieslik (2015)
Genome Res. 25:1372-1381).
[0611] MicroRNA (miRNA). The present disclosure can assess the
influence of a released bead-bound compound on expression profile
of miRNAs in a given cell or, alternatively, on expression profiles
of the population of mRNAs that are specifically bound by a given
species of miRNA (Jain, Ghosh, Barh (2015) Scientific Reports.
5:12832). For example, the present disclosure provides a bead that
contains: (1) Bead-bound compound; (2) Bead-bound DNA barcode; and
(3) Bead-bound response capture element, where the response capture
element either captures miRNA or where the response capture element
includes a species of miRNA (as part of the response capture
element). Expression profiles for microRNA have been found for
various types of cancer, for example, breast cancer breast cancer
(Tanja (2009) J. Clin. Oncol. 27:15 Suppl. 538).
[0612] Methods are available for capturing selected populations of
mRNA from the entire transcriptome. Selectivity can be conferred by
using one type of microRNA, such as miR-34a, as a bridging compound
in a "pull-down" assay. In brief, "The transcripts pulled down with
miR-34a were . . . enriched for their roles in growth factor
signaling and cell cycle progression" (Lal, Thomas, Lieberman
(2011) PLOS Genetics. 7:e1002363). The mRNA molecules that are
captured are those that bind to the miR-34A.
[0613] Further methods for capturing mRNA and analyzing expression
level is available (Bacher (2016) Genome Biology. 17:63; Svensson
(2017) Nature Methods. 14:381; Miao and Zhang (2016) Quantitative
Biol. 4:243; Gardini (2017) Nature Methods. 12:443). Cellular
response taking the form of changes in enhancer RNA can be measured
(see, Rahman (2017) Nucleic Acid Res. 45:3017).
[0614] The present invention is not to be limited by compositions,
reagents, methods, systems, diagnostics, laboratory data, and the
like, of the present disclosure. Also, the present invention is to
not be limited by any preferred embodiments that are disclosed
herein.
Sequence CWU 1
1
1124DNAArtificial SequenceSynthetic Construct 1ctcacatccc
attttcgctt tagt 24
* * * * *