U.S. patent application number 15/452730 was filed with the patent office on 2017-07-06 for monoliths with attached recognition compounds, arrays thereof and uses thereof.
The applicant listed for this patent is DICE Molecules SV, LLC. Invention is credited to Pehr Harbury, Madan Paidhungat, Robin Prince.
Application Number | 20170191056 15/452730 |
Document ID | / |
Family ID | 53678148 |
Filed Date | 2017-07-06 |
United States Patent
Application |
20170191056 |
Kind Code |
A1 |
Harbury; Pehr ; et
al. |
July 6, 2017 |
Monoliths with Attached Recognition Compounds, Arrays Thereof and
Uses Thereof
Abstract
Provided herein are monoliths with attached recognition
compounds which selectively bind ligands, methods of preparing such
monoliths, arrays thereof and uses thereof. For example, monoliths
provide herein can be used in columns and arrays thereof.
Inventors: |
Harbury; Pehr; (Portola
Valley, CA) ; Paidhungat; Madan; (San Francisco,
CA) ; Prince; Robin; (San Carlos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DICE Molecules SV, LLC |
Redwood City |
CA |
US |
|
|
Family ID: |
53678148 |
Appl. No.: |
15/452730 |
Filed: |
March 8, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14608114 |
Jan 28, 2015 |
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15452730 |
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61932747 |
Jan 28, 2014 |
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62006838 |
Jun 2, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 2219/005 20130101;
B01J 2219/00639 20130101; B01J 19/0046 20130101; C40B 50/14
20130101; C12N 15/1093 20130101; C12N 15/1068 20130101; B01J
2219/00423 20130101; B01J 2219/00315 20130101; B01J 2219/00596
20130101; B01J 2219/00572 20130101; C40B 50/16 20130101; B01J
2219/00722 20130101; C12N 15/1065 20130101; B01J 2219/00547
20130101; B01J 2219/00592 20130101; B01J 2219/00509 20130101; B01J
2219/00641 20130101; C40B 70/00 20130101; C12N 15/1068 20130101;
C12Q 2563/179 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; B01J 19/00 20060101 B01J019/00 |
Claims
1-20. (canceled)
21. A method of routing a mixture of k nucleic acids tags, each
nucleic acid tag operatively linked to a chemical reaction site or
ligand, to m spatial locations, where m and k are integers greater
than 1, comprising: contacting the mixture of k nucleic acids with
an array, which comprises m spatial locations where each spatial
location comprises a support with a monolith attached to the
support, wherein each monolith includes a covalently attached
polymer which selectively hybridizes to one of the k nucleic acid
tags; and hybridizing each of the k nucleic acid tags with the
complementary polymer in one of the m spatial locations of the
array.
22. The method of claim 21, further comprising eluting the k
nucleic acid tags from the polymer to m spatially defined
locations.
23. The method of claim 21, wherein the array comprises a block
including m discrete spatial locations.
24. The method of claim 23, wherein the array comprises more than
one operatively linked block.
25. The method of claim 21, wherein the rate constant of
hybridizing the nucleic acid tags with the polymer at the one or
more spatial locations is between about 1.times.10.sup.2
M.sup.-1s.sup.-1 and about 1.times.10.sup.6 M.sup.-1s.sup.-1.
26. The method of claim 21, wherein the yield of hybridizing the
nucleic acid tags with the polymer at the one or more spatial
locations is greater than about 40%.
27. The method of claim 21, wherein the yield of hybridizing the
nucleic acid tags with the polymer at the one or more spatial
locations is greater than about 90%.
28. The method of claim 21, wherein the polymer is a capture
oligonucleotide.
29. The method of claim 28, wherein the capture oligonucleotide
comprises between about 10 nucleotides and about 50 nucleotides in
length, 15 nucleotides and about 45 nucleotides in length, 10
nucleotides and about 30 nucleotides in length, between about 15
nucleotides and about 24 nucleotides in length or between about 19
nucleotides and about 20 nucleotides in length.
30. The method of claim 28, wherein the capture oligonucleotides
are orthogonal.
31. The method of claim 28, wherein the T.sub.m of the capture
oligonucleotides is between about 49.degree. C. and about
53.degree. C.
32. The method of claim 28, wherein k is greater than or equal to
10 and about 40% of the nucleic acid tags are hybridized to
complementary capture oligonucleotides and the number of
complementary capture oligonucleotides is greater than or equal to
10.
33. The method of claim 28, wherein k is greater than 10 and about
90% of the nucleic acid tags are hybridized to complementary
capture oligonucleotides and the number of complementary capture
oligonucleotides is greater than or equal to 10.
34. The method of claim 28, wherein the nucleic acid tags are
hybridized to the capture oligonucleotides at a temperature at or
below about 10.degree. C. of the T.sub.m of the capture
oligonucleotides.
35. The method of claim 21, wherein the support is comprised of
titanium, aluminum alloys, stainless steel, doped metals, glass,
quartz, polycarbonate, fused silica, poly (methyl methacrylate),
plastics, polyether ether ketone, doped polyether ether ketone,
doped polystyrene, cyclic olefin copolymer, polyetheriimide, doped
polypropylene or combinations thereof.
36. The method of claim 21, wherein the array is a series of
columns, which are operatively linked.
37. The method of claim 21, wherein the monolith is an organic
polymer monolith.
38. The method of claim 21, wherein the support binds between about
0.5 fmol/.mu.l and about 0.4 nmol/.mu.l of the nucleic acid
tag.
39. The method of claim 21, wherein the nucleic acid tag comprises
between about 20 nucleotides and about 1000 nucleotides in length,
between about 50 nucleotides and about 800 nucleotides in length,
between about 100 nucleotides and about 600 nucleotides in length,
between about 150 nucleotides and about 300 nucleotides in
length.
40. The method of claim 21, wherein the ligand is a peptide, a
peptoid or an organic compound of molecular weight of between about
50 and about 3000 daltons.
41. The method of claim 21, wherein the monolith is covalently
attached to the support.
42. The method of claim 21, wherein the monolith is covalently
attached to the support through an amide, ester, urea, urethane,
carbon-silicon, carbon-nitrogen, carbon-carbon, ether, thioether,
silicon-oxygen or disulfide bond.
43. A method of routing a mixture of k nucleic acids tags to m
spatial locations, where m and k are integers greater than 1
comprising: contacting the mixture of k nucleic acids with an
array, which comprises m spatial locations where each spatial
location comprises a support, with a monolith attached to the
support, wherein each monolith includes a covalently attached
polymer which selectively hybridizes to one of the k nucleic acid
tags; and hybridizing each of the k nucleic acid with the
complementary polymer in one of the m spatial locations of the
array.
44. A method of preparing a nucleic acid programmed library of
chemical compounds comprising: (a) routing k nucleic acid tags
using the method of claim 21; (b) eluting each nucleic acid tag
from the polymer to m spatially defined locations; (c) reacting the
m spatially localized nucleic acid tags with x different chemical
subunits, wherein x is an integer greater than 1; (d) pooling the k
nucleic acid tags with x different attached chemical subunits; and
(e) repeating steps (a) through (d) y times where y is an integer
greater than 1.
45. The method of claim 44, wherein the nucleic acid tags eluted in
step (b) are immobilized by an anionic exchange resin after step
(b) and are eluted from the anionic exchange resin after step
(c).
46. The method of claim 44, wherein the members of the library are
greater than about 1000 members.
Description
[0001] This application claims priority under 35 U.S.C. .sctn.119
(e) from U.S. Provisional Application Ser. Nos. 61/932,747 and
62/006,845, filed Jan. 28, 2014 and Jun. 2, 2014, respectively, and
is a divisional of U.S. Ser. No. 14/608,114, filed Jan. 28, 2015,
which are hereby incorporated by reference in their entirety.
FIELD
[0002] Provided herein are monoliths with attached recognition
compounds which selectively bind ligands, methods of preparing such
monoliths, arrays thereof and uses thereof. For example, monoliths
provided herein can be used in columns and arrays thereof.
BACKGROUND
[0003] Crosslinked polymer supports have been useful in catalysis,
separations and solid phase synthesis. Crosslinked polymer supports
were initially provided as homogeneous porous particles, which were
typically used in continuous flow processes including, inter alia,
chromatography. However, a number of significant issues exist with
respect to the use of particulate sorbents: slow exchange between
convective flow and binding to the solid support which leads to
poor resolution, large void volume between packed particles, high
back pressures and low dynamic binding capacity, particularly for
macromolecules. The above limitations have restricted the use of
homogeneous porous particles as functionalized supports with
attached recognition molecules which can bind various ligands.
[0004] More recently, porous monolithic materials have been
developed (Arrua, et al., Materials (2009) 2 2429-2466; Svec et
al., Monolith Materials, J. of Chromatography Library, Vol. 67,
Svec et al., (Eds.); Wu et al., J. Chromatography A (2008)
369-392). These heterogeneous macroporous polymers have a rigid
porous structure which is formed during preparation and is usually
maintained in any solvent or in a dry state and imparts a sponge
like quality to the monolith. Importantly, the problems of large
void volumes (i.e., high permeability), slow exchange (i.e., poor
rates of mass transfer) of macromolecules, poor resolution and high
back pressures are mitigated in such monolithic materials where
fluid flow is through the pores of the monolith. Currently,
monoliths have been used mainly for chromatographic separations
with relatively little attention devoted to the preparation of
monoliths functionalized with attached recognition molecules
(particularly, DNA) for ligand binding and use, for example, in
arrays.
[0005] Accordingly, what is needed are monoliths which include
attached recognition compounds and arrays of these monoliths Such
monoliths and arrays thereof will be useful, inter alia, in ligand
binding.
SUMMARY
[0006] The present invention provides monoliths functionalized with
recognition compounds, arrays thereof and uses thereof. In one
aspect, monoliths which have attached recognition compounds are
provided. The recognition compounds selectively bind ligands. In
some embodiments, the recognition compounds are oligonucleotides,
single stranded RNA, single stranded DNA, DNA binding proteins, RNA
binding proteins, peptide nucleic acids, peptides, depsipeptides,
polypeptides, antibodies, peptoids, polymers, polysiloxanes,
inorganic compounds of molecular weight greater that 50 daltons,
organic compounds of molecular weight between about 3000 daltons
and about 50 daltons or combinations thereof. In other embodiments,
the ligands are oligonucleotides, single stranded RNA, single
stranded DNA, DNA binding proteins, RNA binding proteins, peptide
nucleic acids, peptides, depsipeptides, polypeptides, antibodies,
peptoids, polymers, polysiloxanes, inorganic compounds of molecular
weight greater that 50 daltons, organic compounds of molecular
weight between about 3000 daltons and about 50 daltons or
combinations thereof.
[0007] In some embodiments, a housing is provided which includes a
monolith which encompasses attached recognition compounds that
selectively bind ligands. In other embodiments, the housing
selectively binds members of compound libraries, which may be
provided by phage display, RNA display or nucleic acid programmable
combinatorial chemistry.
[0008] In some embodiments, an array is provided. The array
encompasses two or more housings which include a monolith which
includes attached recognition compounds that selectively bind
ligands. In other embodiments, the array includes a block which
encompasses two or more wells which contain housings. The housings
include a monolith which encompasses attached recognition compounds
that selectively bind ligands.
[0009] In still another aspect, a monolith media is provided. The
monolithic media includes aggregated particles with attached
recognition compounds which selectively bind ligands.
[0010] In still another aspect, an array including two or more ion
exchange housings is provided. The ion exchange housings include a
monolith with an ionizable group. In some embodiments, the array
includes filter plates or any other type of microplates or devices
which allow for flow through of the mobile phase and a block which
encompasses two or more wells which contain ion exchange material.
The ion exchange material encompasses a monolith which includes an
ionizable group.
[0011] In yet another aspect, a method for preparing a nucleic acid
programmed library of chemical compounds is provided. The method
encompasses the steps of contacting a mixture of nucleic acid
molecules with an array including a block which has two or more
addressable wells. Each well includes a monolith with one or more
attached recognition compounds which selectively bind single
stranded nucleic acids thereby splitting the nucleic acid molecules
into subpopulations. The subpopulations of nucleic acid molecules
may optionally be dissociated from the recognition compounds,
using, for example, elevated temperature, change in ionic strength
or change in pH with the dissociated nucleic acid molecules
transferred to separate containers. The separated subpopulations of
nucleic acid molecules are then reacted with different chemical
subunits, where the nucleic acid molecules include at least one
binding sequence and one chemical reaction site. When the
subpopulations of nucleic acid molecules are optionally transferred
to separate containers the wells which include monoliths with
attached recognition compounds which selectively bind nucleic acids
are aligned in addressable manner with the separate containers.
[0012] In still another aspect, a method for preparing a nucleic
acid programmed library of chemical compounds is provided. The
method encompasses the steps of contacting a mixture of nucleic
acid molecules with an array including a block which has two or
more addressable wells. Each well includes a monolith with one or
more attached recognition compounds which selectively bind single
stranded nucleic acids thereby splitting the nucleic acid molecules
into subpopulations. The subpopulations of nucleic acid molecules
are transferred to a second array including filter plates or any
other type of microplates or devices which allow for flow through
of the mobile phase and a block containing two or more addressable
wells. The subpopulations of nucleic acid molecules may be
dissociated from the recognition compounds using, for example,
elevated temperature, change in ionic strength or change in pH. The
wells of the second array include anion exchange material which
non-specifically immobilizes the subpopulations of nucleic acid
molecules. The immobilized subpopulations of nucleic acid molecules
are reacted with different chemical subunits. The wells which
include monoliths with one or more attached recognition compounds
which selectively bind nucleic acids are aligned in addressable
manner with the wells including the anion exchange material. The
nucleic acid molecules include at least one binding sequence and
one chemical reaction site. In some embodiments, the anion exchange
material includes a monolith with anion exchange groups.
[0013] In still another aspect, a device is provided. The device
encompasses two arrays which include separate blocks. The block of
the first array encompasses two or more addressable wells which
include monoliths with attached recognition compounds which
selectively bind ligands. The block of the second array includes
filter plates or any other type of microplates or devices which
allow for flow through of the mobile phase and two or more
addressable wells which include ion exchange material. The wells
which include monoliths with attached recognition compounds which
selectively bind ligands are aligned with the wells including the
ion exchange material. In some embodiments, the ion exchange
material includes a monolith with ion exchange groups.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows an exemplary DNA-directed splitting of a
library of fragments. The degenerate family of nucleic acid tags in
this example is composed of catenated 20 base-pair nucleotide
sequences, which are either constant (C.sub.1-C.sub.5) or variable
(a.sub.1-j.sub.4). The letters a.sub.1 through j.sub.4 in the
variable regions of the DNA fragments denote distinct 20 nucleotide
sequences with orthogonal hybridization properties. To carry out
the first split, the degenerate family of fragments is passed over
a set of ten different affinity resins displaying the sequences
a.sub.1.sup.c-j.sub.1.sup.c, which are complementary to the
sequences a.sub.1-j.sub.1 in the first variable region (an
exemplary affinity resin is represented by the circle). Ten
sub-pools of the original family of fragments result. Each sub-pool
of nucleic acid tags is then reacted with a distinct chemical
monomer to allow for coupling of the distinct chemical monomer at
the chemical reaction site of each nucleic acid tag. The sub-pools
are then recombined, and the library is split into a new set of
sub-pools based on the sequences a.sub.2-j.sub.2, etc.
[0015] FIG. 2 shows an exemplary chemical coupling reaction at the
chemical reaction site of a nucleic acid tag. A nucleic acid tag
comprising a chemical reaction site is treated with the NHS ester
of FMOC-alanine in DMF. The FMOC protecting group is removed with
piperidine to provide an alanine coupled to the chemical reaction
site of the nucleic acid tag. The process can be repeated many
times, and with a variety of amino acids at successive steps in
order to produce a library of distinct polypeptides.
[0016] FIGS. 3A-3D illustrate a method of partition based chemical
synthesis using a series of columns to generate a library of
distinct chemical compounds.
[0017] FIG. 4 illustrates an exemplary hybridization array with
A11, D11, T1, D12 and A12 from left to right. Front face is on the
top, and the top of the device is on the left.
[0018] FIG. 5 illustrates an exemplary transfer array with D01, T1
and D02 from left to right. Front face is on the top, and the top
of the device is on the left.
DETAILED DESCRIPTION
Definitions
[0019] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs. In
the event that a plurality of definitions for a term exists, those
in this section prevail unless stated otherwise.
[0020] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an" and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a tag" includes a plurality of such tags and
reference to "the compound" includes reference to one or more
compounds and equivalents thereof known to those skilled in the
art, and so forth.
[0021] It is further noted that the claims may be drafted to
exclude any optional element. As such, this statement is intended
to serve as antecedent basis for use of such exclusive terminology
as "solely", "only" and the like in connection with the recitation
of claim elements, or the use of a "negative" limitation.
[0022] "Alkyl" as used herein means any saturated or unsaturated,
branched or unbranched, cyclised, or combination thereof, typically
having 1-10 carbon atoms, which includes methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl,
isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, which may he
optionally substituted with methyl.
[0023] "Alkylene" as used herein means any branched or unbranched,
cyclised, or combination thereof, typically having 1-10 carbon
atoms, which includes methyl, ethyl, propyl, isopropyl, butyl.,
isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl,
hexyl, isohexyl, cyclohexyl, which may be optionally substituted
with methyl.
[0024] "Amplifying population of compounds" as used herein refers
to an increasing population of compounds synthesized according to
the catenated hybridization sequences of nucleic acid tags produced
by the iterative methods described herein.
[0025] "Antibody" as used herein refers to a protein comprising one
or more polypeptides substantially or partially encoded by
immunoglobulin genes or fragments of immunoglobulin genes, e.g., a
fragment containing one or more complementarity determining region
(CDR). The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as myriad immunoglobulin variable region genes. Light
chains are typically classified as either, e.g., kappa or lambda.
Heavy chains are typically classified e.g., as gamma, mu, alpha,
delta, or epsilon, which in turn define the immunoglobulin classes,
IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin
(antibody) structural unit comprises a tetramer. In nature, each
tetramer is composed of two identical pairs of polypeptide chains,
each pair having one "light" (about 25 kD) and one "heavy" chain
(about 50-70 kD). The N-terminus of each chain defines a variable
region of about 100 to 110 or more amino acids primarily
responsible for antigen recognition. The terms variable light chain
(VL) and variable heavy chain (VH) refer to these light and heavy
chains respectively. Antibodies exist as intact immunoglobulins or
as a number of well characterized fragments produced by digestion
with various peptidases. Thus, for example, pepsin digests an
antibody below the disulfide linkages in the hinge region to
produce F(ab)'2 (fragment antigen binding) and Fc (fragment
crystallizable, or fragment complement binding). F(ab)'2 is a dimer
of Fab, which itself is a light chain joined to VH-CH1 by a
disulfide bond. The F(ab)'2 may be reduced under mild conditions to
break the disulfide linkage in the hinge region thereby converting
the (Fab').sub.2 dimer into a Fab' monomer. The Fab' monomer is
essentially a Fab with part of the hinge region. The Fc portion of
the antibody molecule corresponds largely to the constant region of
the immunoglobulin heavy chain, and is responsible for the
antibody's effector function (see, Fundamental Immunology, 4.sup.th
edition. W. E. Paul, ed., Raven Press, N.Y. (1998), for a more
detailed description of antibody fragments). While various antibody
fragments are defined in terms of the digestion of an intact
antibody, one of skill will appreciate that such Fab' or Fc
fragments may be synthesized de novo either chemically or by
utilizing recombinant DNA methodology, peptide display, or the
like. Thus, the term antibody, as used herein, also includes
antibody fragments either produced by the modification of whole
antibodies or synthesized de novo using recombinant DNA
methodologies. Antibodies also include single-armed composite
monoclonal antibodies, single chain antibodies, including single
chain Fv (sFv) antibodies in which a variable heavy and a variable
light chain are joined together (directly or through a peptide
linker) to form a continuous polypeptide, as well as diabodies,
tribodies, and tetrabodies (Pack et al. (1995) J Mol Biol. 246:28;
Biotechnoi 11:1271; and Biochemistry 31:1579). The antibodies are,
e.g., polyclonal, monoclonal, chimeric, humanized, single chain,
Fab fragments, fragments produced by an Fab expression library, or
the like.
[0026] "Base-specific duplex formation" or "specific hybridization"
as used herein refer to temperature, ionic strength and/or solvent
conditions effective to produce sequence-specific pairing between a
single-stranded oligonucleotide and its complementary-sequence
nucleic acid strand, for a given length oligonucleotide. Such
conditions are preferably stringent enough to prevent or largely
prevent hybridization of two nearly-complementary strands that have
one or more internal base mismatches. In some embodiments, the
region of identity between two sequences forming a base-specific
duplex is greater than about 5 bp. In other embodiments, the region
of identity is greater than 10 bp.
[0027] "Capture nucleic acid", "capture oligonucleotide", and
"immobilized capture nucleic acid" as used herein refer to a
nucleic acid sequence attached to a monolith. In general, the
sequence of a capture nucleic acid is complementary to one of the
different hybridization sequences (e.g., a.sub.1, b.sub.1, c.sub.1,
etc.) of the nucleic acid tags and therefore allows for
sequence-specific splitting of a population of nucleic acid tagged
molecules into a plurality of sub-populations of distinct nucleic
acid tagged molecules in separate containers.
[0028] "Chemical reaction site" as used herein refers to a chemical
component of a nucleic acid tag capable of forming a variety of
chemical bonds including, but not limited to; amide, ester, urea,
urethane, carbon-carbonyl bonds, carbon-nitrogen bonds,
carbon-carbon single bonds, olefin bonds, thioethe bonds, and
disulfide bonds.
[0029] "Combinatorial library" as used herein refers to a library
of molecules containing a large number, typically between 10.sup.3
and 10.sup.15 or more different compounds typically characterized
by different sequences of subunits, or a combination of different
side chains functional groups and linkages.
[0030] "DAEM" as used herein refers to 2(d thylam o)ethyl
methacrylate.
[0031] "DEGDM " as used herein refers to diethylene glycol
dimethacrylate.
[0032] "Depsipeptide" as used herein refers to a peptide as defined
herein where one or more of amide bonds are replaced by ester
bonds.
[0033] "Different-sequence small-molecule compounds" refers to
small organic molecules, typically, but not necessarily, having a
common parent structure, such as a ring structure, and a plurality
of different R group substituents or ring-structure modifications,
each of which takes a variety of forms, e.g., different R groups.
Such compounds are usually non-oligomeric (i.e., do not consist of
sequences of repeating similar subunits) and may be similar in
terms of basic structure and functional groups, but vary in such
aspects as chain length, ring size or number, or patterns of
substitution.
[0034] "EDMA" as used herein refers to ethylene glycol
dimethacrylate.
[0035] "Genetic recombination of nucleic acids tags" as used herein
refers to forming chimeras of nucleic acid tags derived from
compounds having one or more desired activities. Chimeras can be
formed by genetic recombination, after repeated cycles of
enrichment and step-wise synthesis, PCR amplification and step-wise
synthesis, partial digestion, reformation and stepwise synthesis to
yield a highly enriched subpopulation of nucleic acid tags which
are bound to compounds having one or more desired activities.
[0036] "GMA" as used herein refers to glycidyl methacrylate.
[0037] "HEMA" as used herein refers to 2-hydroxyl ethyl
methacrylate.
[0038] "Hydrates" refers to incorporation of water into to the
crystal lattice of a compound described herein, in stoichiometric
proportions, resulting in the formation of an adduct. Methods of
making hydrates include, but are not limited to, storage in an
atmosphere containing water vapor, dosage forms that include water,
or routine pharmaceutical processing steps such as, for example,
crystallization (i.e., from water or mixed aqueous solvents),
lyophilization, wet granulation, aqueous film coating, or spray
drying. Hydrates may also be formed, under certain circumstances,
from crystalline solvates upon exposure to water vapor, or upon
suspension of the anhydrous material in water. Hydrates may also
crystallize in more than one form resulting in hydrate
polymorphism. See e.g., (Guillory, K., Chapter 5, pp. 202-205 in
Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel
Dekker, Inc., New York, N.Y., 1999). The above methods for
preparing hydrates are well within the ambit of those of skill in
the art, are completely conventional and do not require any
experimentation beyond what is typical in the art. Hydrates may be
characterized and/or analyzed by methods well known to those of
skill in the art such as, for example, single crystal X-Ray
diffraction, X-Ray powder diffraction, Polarizing optical
microscopy, thermal microscopy, thermogravimetry, differential
thermal analysis, differential scanning calorimetry, IR
spectroscopy, Raman spectroscopy and NMR spectroscopy. (Brittain,
H., Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical
Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999). In
addition, many commercial companies routinely offer services that
include preparation and/or characterization of hydrates such as,
for example, HOLODIAG, Pharmaparc II, Voie de l'Innovation, 27 100
Val de Reuil, France (http://www.holodiag.com).
[0039] "Ligand" as used herein refers to a oligonucleotide, single
stranded RNA, single stranded DNA, a DNA binding protein, a RNA
binding protein, a peptide nucleic acid, a peptide, a depsipeptide,
a polypeptide, a antibody, a peptoid, a polymer, a polysiloxane, a
inorganic compound of molecular weight greater that 50 daltons, a
organic compound of molecular weight between about 1000 daltons and
about 50 daltons or a combination thereof.
[0040] "Linker" as used herein is any molecule or substance which
performs the function of linking the monolith to the recognition
compound. A linker may vary in structure and length. The linker may
be hydrophobic or hydrophilic, long or short, rigid, semirigid or
flexible, etc. The linking group can comprise, for example, a
polymethylene chain, such as a --(CH.sub.2).sub.n-- chain or a
poly(ethylene glycol) chain, such as a --(CH.sub.2CH.sub.2O).sub.n
chain, where in both cases n is an integer from 1 to about
20,5'-O-Dimethoxytrityl-1,'2'-Dideoxyribose-3'-[(2-cyanoethyl)-(N,N-diiso-
propyl)]-phosphoramidite; 9-O-Dimethoxytrityl-triethylene glycol,
1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;
3-(4,4'-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-ph-
osphoramidite; and 18-O-Dimethoxytritylhexaethyleneglycol,
1,-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,
amino-carboxylic linkers (e.g., peptides (e.g., Z-Gly-Gly-Gly-Osu
or Z-Gly-Gly-Gly-Gly-Gly-Gly-Osu), PEG (e.g., Fmoc-aminoPEG2000-NHS
or amino-PEG (12-24)-NHS), or alkane acid chains (e.g.,
Boc-.epsilon.-aminocaproic acid-Osu)), click chemistry linkers
(e.g., peptides (e.g., azidohomalanine-Gly-Gly-Gly-OSu or
propargylglycine-Gly-Gly-Gly-OSu), PEG (e.g., azido-PEG-NHS), or
alkane acid chains (e.g., 5-azidopentanoic acid,
(S)-2-(azidomethyl)-1-Boc-pyrrolidine, or 4-azido-butan-1-oic acid
N-hydroxysuccinimide ester)), thiol-reactive linkers (e.g., PEG
(e.g., SM(PEG)n NHS-PEG-maleimide), alkane acid chains (e.g.,
3-(pyridin-2-yldisulfanyl)-propionic acid-Osu or sulfosuccinimidyl
6-(3'-[2-pyridyldithio]propionamido)hexanoate))), amidites for
oligonucleotide synthesis (e.g., amino modifiers (e.g.,
6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphora-
midite), thiol modifiers (e.g.,
S-trityl-6-mercaptohexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphorami-
dite, or chick chemistry modifiers (e.g., 5-hexynyl-TTT(T).sub.0-7,
6-hexynyl-TTT(T).sub.0-7,
5-hexyn-1-yl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,
6-hexyn-1-yl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,
3-dimethoxytrityloxy-2-(3-(3-propargyloxypropanamido)propanamido)propyl-1-
-O-succinoyl, long chain alkylamino CPG, or 4-azido-butan-1-oic
acid N-hydroxysuccinimide ester)).
[0041] "Initiator" as used herein refers to any free radical
generator capable of initiating polymerization of monovinyl
monomers or polyvinyl monomers by way of thermal, photo, or redox
initiation."MAA" as used herein refers to methacrylic acid.
[0042] "MAETA" as used herein refers to [2-(methac
loxy)ethyl]trimethyl ammonium chloride.
[0043] ".gamma.-MAPS" as used refers to 3-(trimethoxysilyl) propyl
methacrylate.
[0044] "Monolith" as used herein refers to a continuous stationary
phase (i.e., a single continuous material (e.g., a polymer or
silica base matrix) that contains large interconnected pores or
channels allowing high flow rates of mobile phases at moderate
pressure.
[0045] "Monolithic media" as used herein refers to a packing of
aggregated particles which has significantly lower column pressure
than expected on the basis of particle size.
[0046] "NBE" as used herein refers to norbom-2-ene.
[0047] "Non-specific binding" as used herein with respect to a
"non-specific monolith" refer to binding of nucleic acid that does
not depend on the nucleic acid sequence applied to the monolith.
Exemplary materials for non-specific binding include ion-exchange
materials, which are effective to non-specifically capture nucleic
acid tagged molecules at one ionic strength and release the nucleic
acid tagged molecules, following molecule reaction, at a higher
ionic strength.
[0048] "Nucleic acid" as used herein refers to a oligonucleotide
analog as defined below as well as a double stranded DNA and RNA
molecule. A DNA and RNA molecule may include the various analogs
defined below.
[0049] "Nucleic acid tag-directed synthesis" or "tag-directed
synthesis" or "chemical translation" as used herein refer to
synthesis of a plurality of compounds based on the catenated
hybridization sequences of the nucleic acid tags according to the
methods disclosed herein.
[0050] "Nucleic acid tag", "nucleic acid support",
"synthesis-directing nucleic acid tags", and "DNA-tag" as used
herein mean the nucleic acid sequences which each comprise at least
(i) a different first hybridization sequence, (ii) a different
second hybridization sequence, and (iii) a chemical reaction site.
The "hybridization sequences" refer to oligonucleotides comprising
between about 3 and up to 50, and typically from about 5 to about
30 nucleic acid subunits. Such "nucleic acid tags" are capable of
directing the synthesis of the combinatorial library based on the
catenated hybridization sequences.
[0051] "Oligonucleotides" or "oligos" as used herein refer to
nucleic acid oligomers containing between about 3 and up to about
50, and typically from about 5 to about 30 nucleic acid subunits.
In the context of oligos (e.g., hybridization sequence) which
direct the synthesis of library compounds, the oligos may include
or be composed of naturally-occurring nucleotide residues,
nucleotide analog residues, or other subunits capable of forming
sequence-specific base pairing, when assembled in a linear polymer,
with the proviso that the polymer is capable of providing a
suitable substrate for strand-directed polymerization in the
presence of a polymerase and one or more nucleotide triphosphates,
e.g., conventional deoxyribonucleotides. A "known-sequence oligo"
is an oligo whose nucleic acid sequence is known.
[0052] "Oligonucleotide analog" as used herein refers to a nucleic
acid that has been modified and which is capable of some or all of
the chemical or, biological activities of the oligonucleotide from
which it was derived. An oligonucleotide analog will generally
contain phosphodiester bonds, although in some cases,
oligonucleotide analogs are included that may have alternate
backbones. Modifications of the ribose-phosphate backbone may
facilitate the addition of additional moieties such as labels, or
may be done to increase the stability and half-life of such
molecules. in addition, mixtures of naturally occurring nucleic
acids and analogs can be made. Alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made. The oligonucleotides may be single
stranded or double stranded, as specified, or contain portions of
both double stranded or single stranded sequence. The
oligonucleotide may be DNA, RNA or a hybrid, where the nucleic acid
contains any combination of deoxyribo--and ribo-nucleotides, and
any combination of bases, including uracil, uridine, adenine,
thymine, cytosine, guanine, inosine, xathanine hypoxathanine,
isocytosine, isoguanine, etc.
[0053] "Peptide" as used herein refers to a polymer of amino acid
residues between about 2 and 50 amino acid residues, between about
2 and 20 amino acid residues, or between about 2 and 10 residues.
Peptides include modified peptides such as, for example,
glycopeptides, PEGylated peptides, lipopeptides, peptides
conjugated with organic or inorganic ligands, peptides which
contain peptide bond isosteres (e.g., .psi.[CH.sub.2S],
.psi.[CH.sub.2NH.sub.2], .psi.[COCH.sub.2], .psi.[(E) or (Z)
CH.dbd.CH], etc and also include cyclic peptides. In some
embodiments, the amino acid residues may be any L-.alpha.-amino
acid, D-.alpha.-amino residue, N-alkyl variants thereof or
combinations thereof. In other embodiments, the amino acid residues
may any L-.alpha.-amino acid, D-.alpha.-amino residue, .beta.-amino
acids, .alpha.-amino acids, N-alkyl variants thereof or
combinations thereof.
[0054] "Operatively linked" as used herein refers to at least two
chemical groups or structures that are linked together. For
example, an oligonucleotide may be covalently attached through a
linker to a ligand or a chemical reaction site. In some
embodiments, the groups or structures may remain linked together
through various manipulations, such as, for example, the steps of a
process.
[0055] "Peptide nucleic acid" as used herein refers to
oligonucleotide analogues where the sugar phosphate backbone of
nucleic acids has been replaced by psuedopeptide skeleton (e.g.,
N-(2-aminoethyl)-glycine)(Nielsen et al., U.S. Pat. No. 5,539,082;
Nielsen et al., U.S. Pat. No. 5,773,571; Burchardt et al., U.S.
Pat. No. 6,395,474).
[0056] "Peptoid" as used herein refers to polymers of poly
N-substituted glycine (Simon et al., Proc. Natl. Acad. Sci. (1992)
89(20) 9367-9371) and include cyclic variants thereof.
[0057] "Polypeptide" as used herein refers to a polymer of amino
acid residues typically comprising greater than 50 amino acid
residues and includes cyclic variants thereof. Polypeptide includes
proteins (including modified proteins such as glycoproteins,
PEGylated proteins, lipoproteins, polypeptide conjugates with
organic or inorganic ligands, etc.) receptor, receptor fragments,
enzymes, structural proteins (e.g., collagen) etc. In some
embodiments, the amino acid residues may be any L-.alpha.-amino
acid, D-.alpha.-amino residue, or combinations thereof. In other
embodiments, the amino acid residues may be any L-.alpha.-amino
acid, D-.alpha.-amino residue, N-alkyl variants thereof or
combinations thereof.
[0058] "Polymer" includes copolymer, and the term "monomer"
includes co-monomer. Polymers include, for example, polyamides,
phospholipids, polycarbonates, polysaccharides, polyurethanes,
polyesters, polyureas, polyacetates, polyarylene sulfides,
polyethylenimines, polyimides, etc.
[0059] "Porogen" or "porogenic solvent" as used herein refers to a
solvent capable of forming pores in a polymer matrix during
polymerization thereof, and includes but is not limited to a
aliphatic hydrocarbon, a aromatic hydrocarbon, a ester, a amide, a
alcohol, a ketone, a ether, a solutions of soluble polymer, and a
combination thereof.
[0060] "Recognition Compound" as used herein refers to a
oligonucleotide, single stranded RNA, single stranded DNA, a DNA
binding protein, a RNA binding protein, a peptide nucleic acid, a
peptide, a depsipeptide, a polypeptide, a antibody, a peptoid, a
polymer, a polysiloxanes, a inorganic compounds of molecular weight
greater that 50 daltons, a organic compounds of molecular weight
between about 1000 daltons and about 50 daltons or a combination
thereof.
[0061] "Salt" refers to a salt of a compound, which possesses the
desired pharmacological activity of the parent compound. Such salts
include: (1) acid addition salts, formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid, and the like; or formed with organic acids such as
acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic
acid, glycolic acid, pyruvic acid, lactic acid, malonic acid,
succinic acid, malic acid, maleic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic
acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid,
benzenesulfonic acid, 4-chlorobenzenesulfonic acid,
2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic
acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid,
glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid,
tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid,
glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid,
muconic acid, and the like; or (2) salts formed when an acidic
proton present in the parent compound is replaced by a metal ion,
e.g., an alkali metal ion, an alkaline earth ion, or an aluminum
ion; or coordinates with an organic base such as ethanolamine,
diethanolamine, triethanolamine, N-methylglucamine and the like. In
some embodiments, salts may be formed when an acidic proton present
can react with inorganic bases (e.g., sodium hydroxide, sodium
carbonate, potassium hydroxide, aluminum hydroxide, calcium
hydroxide, etc.) and organic bases (e.g., ethanolamine,
diethanolamine, triethanolamine, tromethamine, N-methylglucamine,
etc.) In some embodiments, the salt is pharmaceutically
acceptable.
[0062] "Solvates" refers to incorporation of solvents into to the
crystal lattice of a compound described herein, in stoichiometric
proportions, resulting in the formation of an adduct. Methods of
making solvates include, but are not limited to, storage in an
atmosphere containing a solvent, dosage forms that include the
solvent, or routine pharmaceutical processing steps such as, for
example, crystallization (i.e., from solvent or mixed solvents)
vapor diffusion, etc.. Solvates may also be formed, under certain
circumstances, from other crystalline solvates or hydrates upon
exposure to the solvent or upon suspension material in solvent.
Solvates may crystallize in more than one form resulting in solvate
polymorphism. See e.g., (Guillory, K., Chapter 5, pp. 205-208 in
Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel
Dekker, Inc., New York, N.Y., 1999)). The above methods for
preparing solvates are well within the ambit of those of skill in
the art, are completely conventional do not require any
experimentation beyond what is typical in the art. Solvates may be
characterized and/or analyzed by methods well known to those of
skill in the art such as, for example, single crystal X-Ray
diffraction, X-Ray powder diffraction, Polarizing optical
microscopy, thermal microscopy, thermogravimetry, differential
thermal analysis, differential scanning calorimetry, IR
spectroscopy, Raman spectroscopy and NMR spectroscopy. (Brittain,
H., Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical
Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999). In
addition, many commercial companies routinely offer services that
include preparation and/or characterization of solvates such as,
for example, HOLODIAG, Pharmaparc II, Voie de l'Innovation, 27 100
Val de Reuil, France (http://www.holodiag.com).
[0063] "Selection for a desired activity" as used herein is any
biochemical procedure that segregates more desirable molecules from
less desirable molecules based on physical properties of the
molecule. As such it included physical segregation of compounds
that exhibit a desired property from a heterogeneous mixture of
molecules. Examples include affinity purification of ligands from
mixtures by binding to an immobilized target protein, or isolation
of enzyme substrates from mixtures by enzyme-mediated attachment of
an affinity handle, etc.
[0064] "SPMA" as used herein refers to sulfopropyl
methacrylate.
[0065] "TRIM" as used herein refers to trimethylolpropane
trimethacrylate.
[0066] "Tagged compounds", "DNA-tagged compound", or "nucleic
acid-tagged compound" are used to refer to compounds containing (a)
unique nucleic acid tags, each unique nucleic acid tag of each
compound includes at least one and preferably two or more catenated
different hybridization sequences, wherein the hybridization
sequences are capable of binding specifically to complementary
immobilized capture nucleic acid sequences, and (b) a chemically
reactive reaction moiety that may include a compound precursor, a
partially synthesized compound, or completed compound. A nucleic
acid tagged compound in which the chemically reactive moiety is a
completed-synthesis compound is also referred to as a nucleic
acid-tagged compound
Monoliths with Attached Recognition Compounds and Arrays
Thereof
[0067] Monoliths are integrated continuous porous media without
interparticular voids which have been typically used as
chromatographic supports (e.g., Ueki et al., Anal. Chem. (2004) 76,
7007-7012; Ueki et al., J. Chromatography A (2006) 1106, 106-111;
Saburadin et al., Analytica Chimica Acta (2012) 736 108-114; Shu et
al., J. Chromatography A (2011) 1218 5288-5234; Lubbad et al., J.
Chromatography A (2011) 1218 8897-8902; Lubbad et al., J.
Chromatography A (2011) 1218 2362-2367). Monoliths, broadly, are
any single bodied structure containing interconnected repeating
cells or channels, that are characterized by a defined porosity and
which support interactions between the solid and surrounding mobile
phase. Mobile phases are forced through the porous monolithic media
which results in convective flow and enhanced mass transfer.
Monoliths may be based on polymers (i.e., organics), silica,
organic-silica hybrids, inorganics, cyrogels and agarose, with the
first two types being the most predominant.
[0068] Provided herein, in the broadest sense, are monoliths with
attached recognition compounds and arrays thereof. In one aspect,
monoliths with attached recognition compounds that selectively bind
ligands are provided. In some embodiments, the monoliths are
porous. In other embodiments, the monoliths include an ionizable
group. In some of these embodiments, the recognition compounds are
ionically attached to the monoliths. In other of these embodiments,
the recognition compounds are covalently attached to the
monoliths.
[0069] In some embodiments, the recognition compounds are
oligonucleotides, single stranded RNA, single stranded DNA, DNA
binding proteins, RNA binding proteins, peptide nucleic acids,
peptides, depsipeptides, polypeptides, antibodies, peptoids,
polymers, polysiloxanes, inorganic compounds of molecular weight
greater that 50 daltons, organic compounds of molecular weight
between about 3000 daltons and about 50 daltons or combinations
thereof. In other embodiments, the ligands are oligonucleotides,
single stranded RNA, single stranded DNA, DNA binding proteins, RNA
binding proteins, peptide nucleic acids, peptides, depsipeptides,
polypeptides, antibodies, peptoids, polymers, polysiloxanes,
inorganic compounds of molecular weight greater that 50 daltons,
organic compounds of molecular weight between about 3000 daltons
and about 50 daltons or combinations thereof. In still other
embodiments, the recognition compounds are oligonucleotides, single
stranded RNA, single stranded DNA, DNA binding proteins, RNA
binding proteins, peptide nucleic acids, peptides, depsipeptides,
polypeptides, antibodies, peptoids, organic compounds of molecular
weight between about 3000 daltons and about 50 daltons or
combinations thereof and the ligands are single stranded DNA,
single stranded RNA, peptides, depsipeptides, polypeptides,
antibodies, peptoids, organic compounds of molecular weight between
about 3000 daltons and about 50 daltons or combinations
thereof.
[0070] In some embodiments, the recognition compounds are
oligonucleotides, single stranded RNA, single stranded DNA, DNA
binding proteins, RNA binding proteins, peptide nucleic acids or
combinations thereof. In other embodiments, the ligands are single
stranded DNA, single stranded RNA or combinations thereof. In still
other embodiments, the recognition compounds are oligonucleotides,
single stranded RNA, single stranded DNA, DNA binding proteins, RNA
binding proteins, peptide nucleic acids or combinations thereof and
the ligands are single stranded DNA, single stranded RNA or
combinations thereof.
[0071] In some embodiments, the recognition compounds are
proteinacious DNA binding proteins such as the lac repressor, trp
repressor, lambda repressor, arc repressor or engineered variants
of these repressors with novel DNA binding specificity and the
ligands are double stranded DNA. In vet other embodiments, the
recognition compounds are site specific nucleases such as CreI
family meganucleases or TALEN nucleases lacking nuclease activity
but retaining sequence specific DNA recognition properties.
[0072] In some embodiments, the monolith is formed from silica
copolymers. In other embodiments, the silica copolymers include a
monomer selected from the group consisting of
##STR00001## ##STR00002##
or combinations thereof.
[0073] Silica based monoliths may be prepared by hydrolysis and
polycondensation of alkoxysilanes, catalyzed by acid, in presence
of a porogen. After heating and drying the sol-gel network may be
derivatized by silation. Functional groups appropriate for
attachment of recognition compounds may be introduced into the
silica monolith by direct incorporation of functionalized monomers
in the fabrication process or by modification of the silica
monolith.
[0074] For example,
##STR00003##
may be functionalized, as described below for GMA-co-EDMA polymer
monoliths. Another silica monolith that may be useful for
practicing the methods described herein is
poly(p-methylstyrene-co-bis(p-vinylbenzyl)dimethsilane (Wieder et
al., J. Chromatography A (2008) 1191 252-263).
[0075] Modification of the silica monolith is usually preferable,
since a change in chemical functionality does not require
optimization of the physical properties of a new monolith. Here,
the sol-gel monoliths are chemically modified by silation with
organochlorosilane or organoalkoxysilane reagents such as some the
monomers shown above. The functional groups on the silica monolith
may be directly functionalized with recognition compounds, for
example, by ether, ester or amide bond formation, if the
recognition compound contains complementary functionality. In some
embodiments, cycloaddition of complementary functional groups
(e.g., azide and acetylene; diene and electron deficient olefin) or
click chemistry (Evans, R. A., Australian J. of Chemistry, 60 (6):
384-395 (2007) may be used to attach the recognition compound to
the monolith.
[0076] Alternatively, a bifunctional linker may be attached to the
functional groups of the silica monolith and the recognition
compound covalently bonded to the monolith through formation of a
amide, carbamate, ester, urea, urethane, carbon-nitrogen,
carbon-carbon, ether, thioether or disulfide bond with a
complementary functional group on the bifunctional linker. In some
embodiments, cycloaddition of complementary functional groups
(e.g., azide and acetylene; diene and electron deficient olefin) or
click chemistry may be used to attach the linker covalently bonded
to the monolith to the recognition compound.
[0077] In addition, the recognition compounds may be functionalized
with a linker, which contains functional groups capable of reacting
with the functional groups on the silica monolith. As before, a
recognition compound attached to a linker may be covalently bonded
to the monolith through formation of an amide, carbamate, ester,
urea, urethane, carbon-nitrogen, carbon-carbon, ether, thioether or
disulfide bond with a complementary functional group on the linker.
In some embodiments, cycloaddition of complementary functional
groups (e.g., azide and acetylene; diene and electron deficient
olefin) or click chemistry may be used to attach the monolith to
the linker covalently bonded to the recognition compound.
[0078] In some embodiments, the monolith is an organic-inorganic
silica hybrid which combines advantages of both inorganic monoliths
and organic monoliths (i. e. , mechanical and structural stability
in organic solvent and easy functionalization). Such monoliths may
be prepared from silane that has been modified to have an organic
functional group as part of its structure.
[0079] In some embodiments, the monolith may be an inorganic
monolith such as, for example, a zirconia, hafnia or titania
monolith Moth et al., J. Chromatogr. A, (2005) 392; Randon et al.,
J. Chromatogr. A, (2006) 19; Rivera et al., Analyst, (2009), 31;
Kubo et al., Mater. Let., (2010) 177; Konishi et al., J.
Chromatiogr. A, (2009) 7375). The above inorganic monoliths are
resistant to extremes of pH and temperature which may be
problematic with silica monoliths and often have unusual and unique
selectivity. In some embodiments, inorganic monoliths may be
functionalized with recognition compounds in the manner described
above for silica monoliths.
[0080] Polymer based monoliths are usually highly crosslinked
structures where the internal structure includes fused arrays
microglobules separated by pores. The structural rigidity of porous
polymer monoliths is due to the extensive crosslinking typically
found in these structures.
[0081] In some embodiments, the monolith comprises organic
copolymers. In other embodiments, the organic copolymer is a
combination of a monovinyl polymers and a polyvinyl polymer. In
still other embodiments, the organic copolymer is a combination of
monovinyl polymers and polyvinyl polymers. In still other
embodiments, the organic copolymer is a polyvinyl polymer or
combinations of polyvinyl polymers.
[0082] In some of the above embodiments, the copolymer includes a
monovinyl monomer selected from the group consisting of vinyl
styrene, vinylnaphthalene, vinylanthracene and their ring
substituted derivatives wherein the substituents include
chloromethyl, alkyls with up to 10 carbon atoms, hydroxyl,
t-butyloxycarbonyl, halogen, nitro, protected hydroxyls or amino
groups, acrylamides, and methacrylamides and their derivatives
substituted on the nitrogen atom with one or two C.sub.1-5 alkyls,
C.sub.1-4 alkylaminoalkyls or dialkylaminoalkyls, C.sub.1-4
methoxyaminoalkyls, C.sub.1-4 dimethoxy or diethoxyaminoalkyls,
C.sub.1-4 methoxyalkyls, tetrahydropyranyl, and tetrahydrofurfuryl
groups, N-acryloylpiperidine, N-acryloylpyrrolidone, and mixtures
thereof, acrylic acid esters, methacrylic acid esters, alkyl
acrylates, alkyl methacrylates, perfluorinated alkyl acrylates,
perfluorinated alkyl methacrylates, glycidyl acrylates, glycidyl
methacrylates, hydroxyalkyl acrylates, hydroxyalkyl methacrylates,
wherein the alkyl group in each of the aforementioned alkyls
consists of 1-10 carbon atoms, sulfoalkyl acrylates, sulfoalkyl
methacrylates, oligoethyleneoxide acrylates, oligoethyleneoxide
methacrylates, acrylate and methacrylate derivatives including
primary, secondary, tertiary, and quarternary amine, epoxide and
zwitterionic functionalities, vinyl pyridines, vinylacetate,
vinylpyrrolidone, vinylazlactone or combinations thereof. In other
embodiments, the monovinyl monomers include, but are not limited
to, styrene, vinylnaphthalene, vinylanthracene and their ring
substituted derivatives wherein the substituents include
chloromethyl, alkyls with up to 18 carbon atoms, hydroxyl,
t-butyloxycarbonyl, halogen, nitro, protected hydroxyls, amino
groups or combinations thereof. In still other embodiments, the
monovinyl monomers include but are not limited to, acrylamides,
methacrylamides and their derivatives substituted on the nitrogen
atom with one or two C.sub.1-5 alkyls, C.sub.1-4 alkylaminoalkyls
or dialkylaminoalkyls, C.sub.1-4 methoxyaminoalkyls, C.sub.1-4
dimethoxy or diethoxyaminoalkyls, C.sub.1-4 methoxyalkyls,
tetrahydropyranyl, and tetrahydrofurfuryl groups,
N-acryloylpiperidine and N-acryloylpyrrolidone or combinations
thereof. In still other embodiments, the monovinyl monomer may also
be selected from the group consisting of acrylic and methacrylic
acid esters, alkyl acrylates, alkyl methacrylates, perfluorinated
alkyl acrylates, perfluorinated alkyl methacrylates, hydroxyalkyl
acrylates, hydroxyalkyl methacrylates, wherein the alkyl group in
each of the aforementioned alkyls consists of 1-10 carbon atoms,
sulfoalkyl acrylates, sulfoalkyl methacrylates, oligoethyleneoxide
acrylates, oligoethyleneoxide methacrylates, and acrylate and
methacrylate derivatives including primary, secondary, tertiary,
and quartemary amine, epoxide and zwitterionic functionalities,
vinylacetate, vinylpyrrolidone, yinylazlactone and combinations
thereof.
[0083] In some embodiments, the copolymer includes a monovinyl
monomer selected from the group consisting of
##STR00004##
and combinations thereof.
[0084] In some embodiments, the polyvinyl monomer is an alkylene
diacrylate, alkylene diacrylamide, alkylene dimethacrylate,
alkylene diacrylamide, alkylene dimethacrylamide, hydroxyalkylene
diacrylate, hydroxyalkylene dimethacrylate, wherein the alkylene
group in each of the aforementioned alkylene monomers consists of
1-10 carbon atoms, oligoethylene glycol diacrylate, oligoethylene
glycol dimethacrylate, vinyl esters of polycarboxylic acid,
divinylbenzene, divinylnaphthalene, pentaerythritol dimethacrylate,
pentaerythritol trirnethacrylate, pentaerythritol
tetramethacrylate, pentaerythritol diacrylate, pentaerythritol
triacrylate, pentaerythritol tetraacrylate, trimethylopropane
trimethacrylate, trimethylopropane acrylates or combinations
thereof. In other embodiments, the polyvinyl monomer is selected
from the group consisting of
##STR00005##
and combinations thereof.
[0085] Polymer monoliths are usually fabricated from a mixture
including a free radical initiator and monomers (including at least
one polyvinyl monomer) dissolved in at least one porogen. Typical
porogenic solvents include common organic solvents, such as, for
example, tetrahydrofuran, acetonitrile, toluene, chlorobenzene,
hexane, methanol, dimethylformamide, cyclohexanol, dodecanol,
supercritical CO.sub.2, ether, etc. Formation of the monolith is
triggered by breakdown of a initiator (e.g., AIBN, TEMPO, APS,
TMED, etc.) into a free radical by an external source (e.g.,
photoinitiation, heat, etc.) or photoexcitation of an initiator
(e.g., benzophenone) which induces the formation of polymer chains
that precipitate out of the reaction mixture eventually
agglomerating together to form a continuous solid structure. The
morphology of the monolith is dependent on numerous variables, such
as, for example, the polyvinyl monomer(s), the monovinyl monomers,
temperature, the composition and percentage of the porogenic
solvents (porogens), the concentration of the free radical
initiator and the method used to initiate polymerization.
[0086] In some embodiments, polymer monoliths are prepared by
polymerizing a mixture which includes one or more polyvinyl
monomers in the presence of an initiator and a porogen. In other
embodiments, polymer monoliths are prepared by polymerizing a
mixture which includes one or more polyvinyl monomers in the
presence of an initiator, a porogen and one or more monovinyl
monomers. The mixture is usually washed with a suitable solvent to
remove the porogen and other impurities.
[0087] In some embodiments, the mixture is one or more polyvinyl
monomers in an amount of about 10 to about 60 vol %, about 45% to
about 90 vol % porogens and between about 0.1 to about 1 vol %
initiator. In other embodiments, the mixture is one or more
polyvinyl monomers in an amount of about 10 to about 50 vol %,
about 45% to about 85 vol % porogens and between about 0.1 to about
1 vol % initiator. In still other embodiments, the mixture is one
or more polyvinyl monomers in an amount of about 20 to 40 vol %,
about 45 to about 80 vol % porogens and between about 0.1 to about
1 vol % initiator. In still other embodiments, the mixture is one
or more polyvinyl monomers in an amount of about 15 to 40 vol %,
about 45 to about 85 vol % porogens and between about 0.1 to about
1 vol % initiator. In still other embodiments, the mixture is about
10-40% of one or more monovinyl monomers, 10 to 40 vol % of one or
more polyvinyl monomers, about 20 to about 80 vol % porogens and
between about 0.1 to about 1 vol % initiator. in still other
embodiments, the mixture is about 20-30% of one or more monovinyl
monomers, 20 to 30 vol % of one or more polyvinyl monomers, about
20 to about 60 vol % porogens and between about 0.1 to about 1 vol
% initiator. In still other embodiments, the mixture is one or
polyvinyl monomers in an amount of about 25 to 35 vol %, about 20
to about 75 vol % porogens and between about 0.1 to about 1 vol %
initiator.
[0088] In some embodiments, the monolith is poly(GMA-co-EDMA),
poly(HEMA-co-EDMA), poly(EDMA-co-MAA), poly(HEMA-co-EDMA-co-SPMA),
poly(GMA-co-DEGDMA), poly(DAEM-co-PEGDA), poly(MAETA-co-PGDA),
poly(HMA-co-EDMA), poly(LMA-co-EDMA), poly(BMA-co-EDMA),
poly(ODMA-co-EDMA), poly(CMS-co-DVB), poly(GMA-co-DVB),
poly(GMA-co-TRIM), poly(styrene-co-DVB),
(NBE-co-(NBE-CH.sub.2O).sub.3SiCH.sub.3). In these embodiments, the
above polymers are made by polymerization of GMA with EDMA, HEMA
with EDMA, EDMA with MAA, HEMA with EDMA and SPMA, GMA with DEGDMA,
DAEM with PEGDA, MAETA with PGDA), HMA with EDMA), LMA with EDMA,
BMA with EDMA, ODMA with EDMA), CMS with DVB, GMA with DVB, GMA
with TRIM and styrene with DVB, respectively.
[0089] Functional groups appropriate for attachment of recognition
compounds may be introduced into the polymer monolith by direct
incorporation of functionalized monomers in the fabrication process
or by modification of the polymer monolith. Some examples of
functionalized monomers include those illustrated below:
##STR00006##
[0090] Modification of polymer monoliths is usually preferable,
since a change in chemical functionality does not require
optimization of the physical properties of a new monolith. Here,
the polymer monoliths may be chemically modified, for example, by
reaction with epoxy, chloromethyl, phenolic hydroxyls and
azalactone functional groups disposed on the surface of the
monolith (Luo et al., J. Chomatogr. A (2001) 926 255; Gusev et al.,
J. Chomatogr. A (1999) 855 273; Xie, et al., Biotechnol Bioeng.
(1999) 62 30) by polymerization of functionalized monomers. The
functional groups on the polymer monolith may be directly reacted
with recognition compounds, for example, by ether, ester or amide
bond formation, if the recognition compound contains complementary
functionality.
[0091] Alternatively, a bifunctional linker may be attached to the
functional groups of the polymer monolith and the recognition
compound covalently bonded to the monolith through formation of a
amide, carbamate, ester, urea, urethane, carbon-nitrogen,
carbon-carbon, ether, thioether or disulfide bond with a
complementary functional group on the bifunctional linker. In some
embodiments, cycloaddition of complementary functional groups
(e.g., azide and acetylene; diene and electron deficient olefin) or
click chemistry may be used to attach the linker covalently bonded
to the monolith to the recognition compound.
[0092] In addition, the recognition compounds may be functionalized
with a linker, which contains functional groups capable of reacting
with the functional groups on the polymer monolith. As before, a
recognition compound attached to a linker may be covalently bonded
to the monolith through formation of an amide, carbamate, ester,
urea, urethane, carbon-nitrogen, carbon-carbon, ether, thioether or
disulfide bond with a complementary functional group on the linker.
In some embodiments, cycloaddition of complementary functional
groups (e.g., azide and acetylene; diene and electron deficient
olefin) or click chemistry may be used to attach the monolith to
the linker covalently bonded to the recognition compound.
[0093] Useful for attachment of recognition groups to polymer
monoliths are monomers which contain oxirane groups, such as, for
example, GMA or derivatives thereof. Copolymers containing such
monomers can be ring opened with various nucleophiles such as, for
example, azide, sulfide ion, amines, etc. which can then be used to
react with complementary functionality on a bifunctional linker, a
linker attached to a recognition compound or a recognition compound
to provide in the last two cases a recognition compound attached to
a monolith. For example, poly(GMA-co-EDMA) or poly(GMA-co-DVB)
after reaction with activated esters, azide, sulfide ion or amines
can react with recognition compounds containing dienes, acetylenes,
thiols and activated esters to provide Diels Alder adducts, 1,3
dipolar cycloadducts, disulfide arid amides, respectively.
Similarly, poly(CMS-co-DVB) after reaction with azide, sulfide ion
or amines can react with recognition compounds containing
acetylenes, thiols and activated esters to provide 1,3 dipolar
cycloadducts, disulfide and amides, respectively. Poly
(GMA-co-EDMA) or poly(GMA-co-DVB) can also be hydrolyzed to diols,
which can then be oxidized to aldehydes. Both of the above
functionalities can react with complementary functionality on a
linker, a linker attached to a recognition compound or a
recognition compound to provide in the last two cases a recognition
compound attached to a monolith.
[0094] In some of the above embodiments, the recognition compound
is a nucleic acid. In other embodiments, the recognition compound
is an oligonucleotide. In exemplary embodiments, poly(GMA-co-EDMA)
or poly(GMA-co-DVB) or poly(CMS-co-DVB) monolith is reacted with
azide to provide a monolith containing an azide functionality. In
some of these embodiments, the oligonucleotides contain a 5'
alkynyl group (e.g., C.sub.3-C.sub.20) which is attached to the 5'
end of the oligonticleotide with a linker (e.g., PEG or poly T). In
some the above embodiments, the oligonucleotide will be linked with
Cu (I) dependent click chemistry to the monolith.
[0095] In other exemplary embodiments, a poly(GMA-co-EDMA) or
poly(GMA-co-DVB) or poly(CMS-co-DVB) monolith is reacted with azide
to provide a monolith containing an azide group. In some of these
embodiments, the oligonucleotides contain terminal alkynes,
dibenzocyclooctyne or bicyclo[6.1.0]nonyne which are attached to
the 5' end of the oligonucleotide with a linker (e.g., PEG or poly
T). In some the above embodiments, the oligonucleotide will be
linked to the monolith with Cu free click chemistry.
[0096] Free radical addition, grafting and photografting approaches
may also be used to functionalize monoliths with reactive
functionality by adding polymeric ligands onto the surface of the
monolith (Myer et al., Macromolecules (2000) 3, 7769-7775; Rohr et
al., Macromolecules (2003) 36, 1677-1684; Wang et al., J.
Chromatography A (2007) 1147, 24-29). Such ligand containing
polymers may substantially increase the density of functional
groups on the monolith surface thus increasing the binding capacity
of the monolith surface. The skilled artisan will appreciate that
many other methods may be used to functionalize polymer monoliths
and attach recognition elements to a monolith.
[0097] In some embodiments, poly(GMA-co-EDMA) or poly(GMA-co-DVB)
after reaction with activated esters, azide, sulfide ion or amines
are converted to ion exchange resins. In other embodiments,
poly(CMS-co-DVB) after reaction with azide, sulfide ion or amines
are converted to ion exchange resins. Poly (GMA-co-EDMA) or
poly(GMA-co-DVB) can also be hydrolyzed to diols, which can then be
oxidized to aldehydes and further converted to ion exchange resins
through functional group manipulation.
[0098] In some embodiments, the porous monolith has a percent
porosity of between about 45% to about 85%. In other embodiments,
the porous monolith has a percent porosity of between about 60% and
about 75%. In still other embodiments, the volume fraction of
mesopores (5 nm-50 nm) is between about 30% and about 80%. In still
other embodiments, the volume fraction of micropores (2 nm) is
between about 0% and about 10%. In still other embodiments, the
volume fraction of pores (50 nm-300 nm) is between about 1% and
about 20%. In still other embodiments, the volume fraction of flow
through pores (>300 nm) is less than about 40%.
[0099] In some embodiments, the pore size of the porous polymer
monolith is in the range of between about 5 nm to about 10,000 inn.
In other embodiments, the pore size of the porous polymer monolith
is in the range of between about 50 nm to about 5,000 nm. In still
other embodiments, the pore size of the porous polymer monolith is
in the range of between about 100 nm to about 10,000 nm. In still
other embodiments, the pore size of the porous polymer monolith is
in the range cif between about 50 nm to about 700 nm.
[0100] In still other embodiments, the average micropore size of
the monolith is less than about 2 nm. In still other embodiments,
the average mesopore size of the monolith is between about 2 nm and
about 50 nm. In still other embodiments, the average micropore size
of the monolith is less than about 2 nm, the average mesopore size
of the monolith is between about 2 nm and about 50 nm and the
average pore size of the monolith is between about 50 nm and about
700 nm.
[0101] In some embodiments, the specific surface area of the
polymer matrix is in the range of between about 0.5 m.sup.2/g to
about 1000 m.sup.2/g. In other embodiments, the specific surface
area of the polymer matrix is in the range of between about 1
m.sup.2/g to about 500 m.sup.2/g. In still other embodiments, the
specific surface area of the polymer matrix is in the range of
between about 5 m.sup.2/g to about 200 m.sup.2/g. In still other
embodiments, the specific surface area of the polymer matrix is in
the range of between about 10 m.sup.2/g to about 100 m.sup.2/g. In
still other embodiments, the specific surface area of the polymer
matrix is in the range of between about 20 m.sup.2/g to about 60
m.sup.2/g, In still other embodiments, the specific surface area of
the polymer matrix is in the range of between about 30 m.sup.2/g to
about 50 m.sup.2/g.
[0102] In some embodiments, the permeability of the monolith is
between about 1 millidarcy and about 1.times.10.sup.3 darcy. In
other embodiments, the permeability of the monolith is between
about 8.9.times.10.sup.2 darcy and about 8.9.times.10.sup.4 darcy.
In still other embodiments, the permeability of the monolith is
between about 1 millidarcy arid about 1.times.10.sup.3 darcy. In
still other embodiments, the permeability of the monolith is about
8.9.times.10.sup.3 darcy.
[0103] While not desiring to be bound by theory, properties of
monoliths which may be important in routing or binding ligands
include rapid hybridization kinetics of large ligand macromolecules
in solution to recognition elements immobilized on the monolith,
low back pressure and high binding capacity of the monolith with
attached recognition element for the ligand.
[0104] In some embodiments, the density of the recognition compound
is between about 1 pmol/10 .mu.l and about 1 .mu.mol/10 .mu.l. In
still other embodiments, the density of the recognition compounds
is about 1 nmol/10 .mu.l.
[0105] In some embodiment, the recognition compounds are
oligonucleotides and the ligands are single stranded DNA, single
stranded RNA sequences or combinations thereof. In some of the
above embodiments, the oligonucleotides have between about 10
nucleic acid subunits and about 50 nucleic acid subunits. In other
of the above embodiments, the oligonucleotides have between about
15 nucleic acid subunits and about 40 nucleic acid subunits. In
still other of the above embodiments, the rate constant of binding
to complementary nucleic acid sequences of the recognition
compounds is between about 1.times.10.sup.2 M.sup.-1s.sup.-1 and
about 1.times.10.sup.6 M.sup.-1s.sup.-1. In still other of the
above embodiments, the rate constant of binding to complementary
nucleic acid sequences of the recognition compounds is between
about 1'10.sup.3 M.sup.-1s.sup.-1 and about 1.times.10.sup.6
M.sup.-1s.sup.-1. In still other of the above embodiments, the rate
constant of binding to complementary nucleic acid sequences of the
recognition compounds is between about 1.times.10.sup.2
M.sup.-1s.sup.-1 and about 1.times.10.sup.5 M.sup.-1s.sup.-1. In
general, the flow through monoliths, such as those describe above
may be useful for rapid and specific DNA/RNA/nucleic acid
hybridization. In some embodiments, the nucleic acid is not a
homopolymer.
[0106] In some embodiments, the monolith is a cryogel monolith
(Malik et al., J. Sep Sci. (2006) 1686; Galaer et al., J. Sep Sci.
(2012) 1173; Arvidsson et al., J. Chromatography A (2002) 27;
Daniak et al., J. Chromatography B (2006) 145. Cryogels are gel
matrices formed in the presence moderately frozen solutions of
monomeric and polymeric precursors which have exemplary chemical
and physical stability. Cryogel monoliths which are typically
polyacrylamide based, possess pores which are typically larger than
those of other gels which make them particularly useful matrices
for large entities such as protein aggregates, membrane fragments,
viruses etc. In some embodiments, recognition compounds may be
attached may be attached to cyrogel monoliths by the methods
provided, supra.
[0107] In some embodiments, the monolith is an agarose based
monolith. In other embodiments, the monolith is a superporous
agarose based monolith. In some of these embodiments, the diameter
of the superpore is between about 20 um and about 200 .mu.m. In
others of these embodiments, the volume of the superpore varies
between about 20% and 50%. Agarose monoliths may be prepared by
casting agarose emulsions (Gustaysson et al., J. Chromatography A,
(1999), 832 29-39; Gustaysson et al., J. Chromatography A, (2000),
925 69-78).
[0108] In general, monoliths derived from agarose can be
functionalized with recognition compounds by reaction with the free
hydroxyl groups of the alternating D-galactose and
L-galactopyranose subunits of the polysaccharide. The hydroxyl
groups may be directly functionalized with recognition compounds,
for example, by ether, ester or carbamate bond formation if the
recognition compound contains complementary functionality.
Alternatively, a bifunctional linker may attached to the hydroxyl
groups of the polysaccharide, and the recognition compound attached
through formation of a amide, carbamate, ester, urea, urethane,
carbon-nitrogen, carbon-carbon, ether, thioether or disulfide bond
or by cycloaddition with an appropriate functional group on the
attached linker (e.g., an azide, diene or electron deficient
olefin) or click chemistry. In addition, the recognition compounds
may be functionalized with a bifunctional linker, which contains a
group capable of reacting with a hydroxyl compound.
[0109] In another aspect, a monolith media is provided. The
monolithic media includes aggregated particles with attached
recognition compounds which selectively bind ligands. By way of
illustration, but without limitation, negatively charged resin
particles may be grafted to provide a positively charged grafted
resin particles. The positively charged grafted resin particles are
then mixed with uncoated resin particles to form aggregated
particles through ionic binding. Recognition compounds can be
attached to aggregated particles by a variety of method know to
those of skill in the art and the aggregated particles can be used
to form a column. The advantage of such an approach is that
covalent or non-covalent adhesion of the monolith to the wall is
not necessary since packing of the aggregated particles prevents
gaps between the wall surface and the monolith.
[0110] In some embodiments, a housing (e.g., a column or well) is
provided. The housing encompasses one or more monoliths which
include attached recognition compounds which selectively bind
ligands. In some embodiments, the monolith is bonded to the
housing. In some embodiments, the housing selectively binds members
of compound libraries. In some of these embodiments, the library is
provided by phage display, RNA display or nucleic acid programmable
combinatorial chemistry. In other of these embodiments, the library
comprises single stranded DNA, single stranded RNA sequences,
peptides, depsipeptides, polypeptides, antibodies, peptoids,
organic compounds of molecular weight between about 3000 daltons
and about 50 daltons or combinations thereof.
[0111] In some embodiments, a housing (e.g., a column or well) is
provided where the recognition compounds are oligonucleotides,
single stranded RNA, single stranded DNA, DNA binding proteins, RNA
binding proteins, peptide nucleic acids, peptides, depsipeptides,
polypeptides, antibodies, peptoids, organic compounds of molecular
weight between about 3000 daltons and about 50 daltons or
combinations thereof and the ligands are single stranded DNA,
single stranded RNA, peptides, depsipeptides, polypeptides,
antibodies, peptoids, organic compounds of molecular weight between
about 3000 daltons and about 50 daltons or combinations thereof. In
other embodiments, the recognition compounds are oligonucleotides,
single stranded RNA, single stranded DNA, DNA binding proteins, RNA
binding proteins, peptide nucleic acids or combinations thereof and
the ligands are single stranded DNA, single stranded RNA or
combinations thereof.
[0112] In some embodiments, a housing (e.g., a column or well) is
provided where the recognition compounds are oligonucleotides and
the ligands are single stranded DNA, single stranded RNA sequences
or combinations thereof. In some of these embodiments, the housing
can bind between about 0.5 fmol/.mu.l and about 0.4 nmol/.mu.l of
single stranded nucleic acid. In other of these embodiments, the
oligonucleotides are sequence specific for the single stranded
nucleic acids. In some of these embodiments, the column routes DNA
libraries, which include attached ligands such as, for example,
peptides, cyclic peptides, triazenes and small organic
molecules.
[0113] In some embodiments, a housing (e.g., a column or well) is
provided which includes a monolith with an attached oligonucleotide
which selectively binds a complementary oligonucleotide operatively
linked to a chemical reaction site or a ligand. In other
embodiments, the oligonucleotide is between about 10 nucleic acid
subunits and about 50 nucleic acid subunits. In still other
embodiments, the oligonucleotide is between about 15 nucleic acid
subunits and about 40 nucleic acid subunits. In still other
embodiments, the rate constant of binding to the complementary
oligonucleotide is between about 1.times.10.sup.2 M.sup.-1s.sup.-1
and about 1.times.10.sup.6 M.sup.-1s.sup.-1. In still other
embodiments, the rate constant of binding to the complementary
oligonucleotide is between about 1.times.10.sup.3 M.sup.-1s.sup.-1
and about 1.times.10.sup.6 M.sup.-1s.sup.-1. In still other
embodiments, the rate constant of binding to the complementary
oligonucleotide is between about 1.times.10.sup.2 M.sup.-1s.sup.-1
and about 1.times.10.sup.5 M.sup.-1s.sup.-1. In still other
embodiments, the complementary oligonucleotide is operatively
linked to a ligand including a chemical reaction site, where the
ligand is a peptide, a peptoid, an organic compound of molecular
weight of less than 2000 daltons.
[0114] In some embodiments, the permeability of the monolith is
between about 1 millidarcy and about 1.times.10.sup.4 darcy. In
other embodiments, the permeability of the monolith is between
about 1 millidarcy and about 1.times.10.sup.3 darcy. In still other
embodiments, the permeability of the monolith is between about
8.9.times.10.sup.2 darcy and about 8.9.times.10.sup.4 darcy. In
still other embodiments, the permeability of the monolith is about
8.9.times.10.sup.3 darcy. In still other embodiments, the column
binds the complementary oligonucleotide preparatively. In still
other embodiments, the column binds between about 0.5 fmol/.mu.l
and between about 0.4 nmol/.mu.l of the complementary
oligonucleotide. In still other embodiments, the oligonucleotide is
attached to the monolith by cycloaddition of an azide group on the
monolith with an alkyne group operatively linked to the
oligonucleotide to form a 1, 2, 3 triazole.
[0115] In some embodiments, an array including two or more columns
which include a monolith with an attached oligonucleotide which
selectively binds a complementary oligonucleotide operatively
linked to a chemical reaction site or a ligand is provided where
the complementary nucleotide is a component of a mixture. In other
embodiments. the array is a block including two or more wells,
where each well includes a different column. In other embodiments,
columns are attached to the surface(s) of the wells. In still other
embodiments, the columns are covalently attached to the surface of
the wells by formation of an amide, ester, urea, urethane,
carbon-silicon, carbon-nitrogen, carbon-carbon, ether, thioether,
or disulfide bond or by cycloaddition. In still other embodiments,
the block is comprised of titanium, aluminum, stainless steel,
doped metals, glass, quartz, polycarbonate, fused silica,
poly(methyl methacrylate), plastics, polyether ether ketone, doped
polyether ether ketone, doped polystyrene, cyclic olefin copolymer,
ultempolyetheriimide, doped polypropylene or combinations thereof.
In still other embodiments, the inner wall of the block is modified
to increase the surface area of the wall. In still other
embodiments, the wells are abraded. In still other embodiments, the
wells are threaded. in still other embodiments, the dimensions of
the well are about 3.5 to 4 mm inner diameter and about 1 to 4 mm
in height. In still other embodiments, the wells are
addressable.
[0116] In some embodiments, an array is provided which includes a
block with two or more wells. In some embodiments, the wells
contain the housings which include attached recognition compounds
which selectively bind ligands. In other embodiments, the monoliths
are attached to the surfaces of the wells. The monoliths may be
covalently attached to the surface of the well by formation of an
amide, ester, urea, urethane, carbon-silicon, carbon-nitrogen,
carbon-carbon, ether, thioether, or disulfide bond to the well
surface. Alternatively, click chemistry or cycloaddition may
provide a cycloadduct between appropriate functionality on the well
surface and groups on the monolith. Functionality may be attached
to the well surface, for example, by silation of the well surface
with a functionalized silane compound (e.g.,
3-trimethoxysilyl)propylmethacrylate) or by ionic attachment of
methacryloyloxydecyldihyrogen phosphate. Alternatively the well
surface may be coated with a polymer (e.g., poly(methyl
methacrylate), polydimethylsiloxane, polyethylene, polypropylene,
poly-2-norborene-co-ethylene) and attached to the monolith via free
radical addition, initiated, for example, by irradiation of
benzophenone. In other embodiments, polyvinyl monomers may be
attached to polymer coated well surfaces during monolith formation
and may further react with pendant olefins on the monolith surface
to covalently bond the monolith to the well surface. In still other
embodiments, the monoliths may be directly attached to the
well.
[0117] In some embodiments, the block comprises titanium, aluminum,
stainless steel, doped metals, glass, quartz, polycarbonate, fused
silica, poly(methyl methacrylate), plastics, polyether ether
ketone, doped polyether ether ketone, doped polystyrene,
ultempolyetheriimide, cyclic olefin copolymer, doped polypropylene
or combinations thereof. The wells of the block may be modified to
increase the surface area of the wall by abrasion, threading or
other methods known to the skilled artisan. In some embodiments,
the dimensions of the well are between about 0.1 mm and about 50 mm
diameter and between about 0.1 mm height and about 10 mm height. In
other embodiments, the dimensions of the well are about 10 mm
diameter and about 10 mm height. In still other embodiments, the
dimensions of the well are about 3.5 to about 4 mm inner diameter
and about 1 to about 4 mm height. In some embodiments, the wells
are addressable.
[0118] In some embodiments an array with two or more ion exchange
housings (e.g., a column or well) is provided which includes filter
plates or any other type of microplates or devices which allow for
flow through of the mobile phase. In some of these embodiments, the
ion exchange housing includes a monolith with an ionizable group.
In some of these embodiments, the ionizable group is an amine, a
carboxylic acid or a sulfonic acid. In some of these embodiments,
the monolith is the reaction product of a copolymer which includes
glycidyl methacrylate with an amine or a sulfonic acid equivalent.
In other of these embodiments, the monolith is the reaction product
of poly(GMA-co-EDMA), poly(GMA-co-DEGDMA) or poly(GMA-co-DVB) with
an amine or sulfonic acid equivalent. In still other of these
embodiments, the monolith is the reaction product of
poiy(CMS-co-DVB) with an amine or sulfonic acid equivalent. In some
embodiments, the ion exchange column includes conventional ion
exchange material.
[0119] In some embodiments, an array is provided which includes
filter plates or any other type of microplates or devices which
allow for flow through of the mobile phase. The array also includes
a block with two or more wells. Each well contains ion exchange
material. In some embodiments, the ion exchange material includes a
monolith with an ionizable group. In some of these embodiments, the
ionizable group is an amine, a carboxylic acid or a sulfonic acid.
In some of these embodiments, the monolith is the reaction product
of a copolymer which includes glycidyl methacrylate with an amine
or a sulfonic acid equivalent. In other of these embodiments, the
monolith is the reaction product of poly(GMA-co-EDMA),
poly(CMS-co-DEGDMA) or poly(GMA-co-DVB) with an amine or sulfonic
acid equivalent. In still other of these embodiments, the monolith
is the reaction product of poly(CMS-co-DVB) with an amine or
sulfonic acid equivalent. In some embodiments, the ion exchange
material is conventional ion exchange material.
[0120] In some embodiments, the columns are attached to the
surface(s) of the wells. In other embodiments, the columns are
covalently attached to the surface of the wells by formation of an
amide, ester, urea, urethane, carbon-silicon, carbon-nitrogen,
carbon-carbon, ether, thioether, or disulfide bond or by
cycloaddition.
[0121] In some embodiments, the block includes titanium, aluminum,
stainless steel, doped metals, glass, quartz, polycarbonate, fused
silica, poly(methyl methacrylate), plastics, polyether ether
ketone, doped polyether ether ketone, doped polystyrene, cyclic
olefin copolymer, ultempolyetheriimide, glass fiber filterplates,
doped polypropylene or combinations thereof.
[0122] In some embodiments, the dimensions of the well are between
about 0.1 mm and about 50 mm diameter and between about 0.1 mm
height and about 10 mm height. In other embodiments, the dimensions
of the well are about 10 mm diameter and about 10 mm height. In
still other embodiments, the dimensions of the well are about 3.5
to 4 mm inner diameter and about 1 to 4 mm height. In some
embodiments, the wells are addressable.
Methods of Using Monoliths with Attached Recognition Compounds and
Arrays Thereof
[0123] A method for preparing a nucleic acid programmed library of
chemical compounds is provided. The method encompasses the steps of
contacting a mixture of nucleic acid molecules with an array which
includes a block which has two or more addressable wells. Each well
includes a monolith with one or more attached recognition compounds
which selectively bind single stranded nucleic acids thereby
splitting the nucleic acid molecules into subpopulations. In some
embodiments, the array includes two or more oligonucleotides where
the attached oligonucleotide of each column selectively binds the
complementary oligonucleotide operatively linked to a chemical
reaction site or a ligand, where the complementary nucleotide is a
component of a mixture.
[0124] The subpopulations of nucleic acid molecules may optionally
be dissociated from the recognition compounds using, for example,
elevated temperature, change in ionic strength or change in pH with
the dissociated nucleic acid molecules transferred to separate
containers. The separated subpopulations of nucleic acid molecules
are then reacted with different chemical subunits, where the
nucleic acid molecules include at least one binding sequence and
one chemical reaction site which are operatively linked. When the
subpopulations of nucleic acid molecules are optionally transferred
to separate containers the wells which include monoliths with
attached recognition compounds which selectively bind nucleic acids
are aligned in addressable manner with the separate containers. In
some embodiments, the separated subpopulations of nucleic acid
molecules are immobilized prior to reaction with different chemical
subunits. In other embodiments, the separated subpopulations of
nucleic acid molecules are immobilized on anion exchange columns
prior to reaction with different chemical subunits. In still other
embodiments, the anion exchange columns include a monolith with an
ion exchange group.
[0125] Another method for preparing a nucleic acid programmed
library of chemical compounds is provided. The method encompasses
the steps of contacting a mixture of nucleic acid molecules with an
array which includes a block which has two or more addressable
wells. Each well includes a monolith with one or more attached
recognition compounds which selectively bind single stranded
nucleic acids thereby splitting the nucleic acid molecules into
subpopulations. In some embodiments, the array includes two or more
oligonucleotides where the attached oligonucleotide of each column
selectively binds the complementary oligonucleotide operatively
linked to a chemical reaction site or a ligand, where the
complementary nucleotide is a component of a mixture.
[0126] The subpopulations of nucleic acid molecules is transferred
to an second array which includes filter plates or any other type
of microplates or devices which allow for flow through of the
mobile phase and a block containing two or more addressable wells.
The subpopulations of nucleic acid molecules may be dissociated
from the recognition compounds, for example, using elevated
temperature change ionic strength or change in pH. The wells of the
second array include anion exchange material which non-specifically
immobilizes the subpopulations of nucleic acid molecules. The
immobilized subpopulations of nucleic acid molecules are reacted
with different chemical subunits. The wells which include monoliths
with one or more attached recognition compounds which selectively
bind nucleic acids are aligned in addressable manner with the wells
including the anion exchange material. The nucleic acid molecules
include at least one binding sequence and one chemical reaction
site which are operatively linked. In some embodiments, the anion
exchange material includes a monolith with anion exchange groups.
In some embodiments, the array includes two or more
oligonucleotides where the attached oligonucleotide of each column
selectively binds the complementary oligonucleotide operatively
linked to a chemical reaction site or a ligand, where the
complementary nucleotide is a component of a mixture. In other
embodiments, the anion exchange material includes a monolith column
with an ion exchange group. In other embodiments, the separated
subpopulations of nucleic acid molecules are immobilized prior to
reaction with different chemical subunits. In still other
embodiments, the separated subpopulations of nucleic acid molecules
are immobilized on anion exchange material prior to reaction with
different chemical subunits.
[0127] As such, the above disclosure represents novel methods for
performing DNA-programmed combinatorial chemistry ("DPCC") (see
e.g., Wrenn et al., J. Am. Chem. Soc. (2007) 129(43) 13137-13143;
Wrenn et al., Annu. Rev. Biochem. (2007) 76, 331-349; Harbury et
al., U.S. Pat. No. 7,479, 472; Harbury et al., U.S. Patent
Application No. US2006/0099626) which is described below.
[0128] DPCC provides methods for synthesizing, screening, and
amplifying a nucleic acid-templated combinatorial chemical library.
The combinatorial chemical library comprise a plurality of species
of bifunctional molecules (i.e., nucleic acid tagged molecules)
that each comprise a different chemical compound moiety and a
unique identifier nucleic acid sequence moiety (i.e., nucleic acid
tag), wherein the nucleic acid sequence defines and directs the
synthesis of the corresponding chemical compound moiety. Details of
the nucleic acid tagged molecules used and traditional strategies
for synthesizing and screening combinatorial nucleic acid tagged
compounds are described in the references above.
[0129] Described below greater detail are nucleic-acid tagged
molecules used for producing small-molecule combinatorial
libraries. Nucleic acid tagged molecules are tagged compounds
having a nucleic acid tag containing at least one, typically two or
more different atenated hybridization sequences and an attached,
typically a covalently attached, chemical reaction moiety (FIG. 1).
The hybridization sequences in any given nucleic acid tag generally
differ from the sequences in any other nucleic acid tag. It should
be noted that different nucleic acid tags can share a common codon.
The hybridization sequences of each nucleic acid tag identify the
particular chemical monomers that will be used in each successive
synthesis step for synthesizing a unique chemical compound attached
to the chemical reaction site. As such, hybridization sequences of
each nucleic acid tag also identify the order of attachment of the
particular chemical monomers to the chemical reaction site.
[0130] In general, each hybridization sequence of the nucleic acid
tag provides a separate sequence for hybridizing to a complementary
capture nucleic acid sequence attached to a monolith. The different
hybridization sequences of the nucleic acid tags allow for
sequence-specific splitting of a population of nucleic acid tagged
molecules into a plurality of sub-populations of distinct nucleic
acid tagged molecules. Each sub-population of nucleic acid tagged
molecules is then reacted with distinct chemical monomer to allow
for coupling of the distinct chemical monomer at the chemical
reaction site of each nucleic acid tag.
[0131] In some embodiments, a set of orthogonal 20-mers (see
orthogonal rule below) that contain Bsal site and have T.sub.m in
the 57-60.degree. C. range are selected. The above are the constant
regions and there are 22 such oligos. The set above is used as a
seed to generate a set of orthogonal 20-mers that have T.sub.m in
the 49-53 range to be used as codons. Orthogonality between a query
and the subject (sequence in seed) is defined by the following
criteria: (a) in-register alignment (query nucleotide 1 matched
with subject nucleotide 1) should have no more than 12 nucleotide s
matching; (b) contiguous runs of not more than 9 nucleotides should
match between query and subject (any register); (c) contiguous runs
of not more than 6 nucleotides matching between query and subject
at either the 3' or 5' end; and (4) all above conditions must be
fulfilled by the reverse complement of the query.
[0132] The set of 20-mers is generated by the following rules: (a)
calculating T. using nearest neighbor method; (b) discard if not in
range (49-53 for codons; 57-61 for constant regions); (c) no
palindromes >4 bps; and (d) no runs of a single base
>4nts.
[0133] To carry out a first reaction step, the population of
nucleic acid tags is "split" into a plurality of sub-populations of
distinct nucleic acid tags, e.g., 10 different sub-populations
corresponding to the ten different hybridization sequences at the
"first" position (V.sub.1, e.g., a.sub.1, b.sub.1, or c.sub.1) in
each tag (FIG. 3A, top and middle panels). This is done by
contacting the nucleic acid tag-containing molecules with a first
group of monoliths with attached capture nucleic acids with
sequences complementary to one of the different "first-position"
hybridization sequences in the nucleic acid tags (e.g., a.sub.1',
b.sub.1', or c.sub.1'). These immobilized nucleic acids are
sometimes referred to herein as "capture nucleic acid" or "capture
oligonucleotides", and the sequences complementary to a nucleic
acid tag sequence referred to as "capture sequences". This
contacting step provides for dividing a population of molecules
having different nucleic acid tags into X.sub.1 sub-populations
(where X represents the number of different capture sequences used
to separate the pooled compounds), where each sub-population of
molecules shares at least one common hybridization sequence within
the nucleic acid tag.
[0134] After the first splitting step, the X.sub.1 different
nucleic acid tag sub-populations, (e.g., ten different
sub-populations of nucleic acid tags as exemplified in FIG. 3A) are
reacted with X.sub.1 different chemical monomers (FIG. 3A, middle
panel). The reactions are performed such that the identity of each
chemical monomer used in the coupling step is directed by the
particular "first" position hybridization sequence of the nucleic
acid tag in the sub-population. As exemplified in FIG. 3A, the
chemical monomer A.sub.1, B.sub.1, or C.sub.1 corresponds to the
particular nucleic acid tag hybridization sequence in the "first"
position (e.g., a.sub.1, b.sub.1, or c.sub.1). The first chemical
coupling step converts the chemical reaction site in each tag to a
reagent--specific compound intermediate, by conjugating the
particular chemical monomer to the chemical reaction site of each
nucleic acid tag sub-population (e.g., A.sub.1, B.sub.1, or
C.sub.1, as exemplified in HG. 2). The result is N.sub.1 different
sub-populations of compounds having nucleic acid tags, each
sub-population having a different chemical monomer conjugated to
the chemical reaction site of each nucleic acid tag sub-population
(FIG. 3A, bottom panel). For example, three different populations
of nucleic acid tags (as separated by hybridization to a.sub.1,
b.sub.1, or c.sub.1 in the "split" step) are represented in the
bottom panel of FIG. 3A, where a first sub-population of molecules
separated by the a.sub.1 sequence is modified to contain the
chemical monomer A.sub.1, a second sub-population of molecules
separated by the b.sub.1 sequence is modified to contain the
chemical monomer B.sub.1, and a third sub-population of molecules
separated by the c.sub.1 sequence is modified to contain the
chemical monomer C.sub.1. In each instance, a chemical monomer is
coupled to the chemical reaction site of the nucleic acid
tag-containing compound, where the added chemical monomer provides
the reaction site for coupling of an additional monomer in a
subsequent step as desired.
[0135] Following the first splitting and chemical coupling steps,
the X.sub.1 different nucleic acid tag-containing compound
sub-populations are pooled and contacted with a second group of
solid-phase reagents (immobilized capture nucleic acid sequences,
e.g., a.sub.2', b.sub.2', or c.sub.2'), each having a sequence that
is complementary to one of the X.sub.2 different "second-position"
hybridization sequences of the nucleic acid tags (e.g., a.sub.2,
b.sub.2, or c.sub.2) (FIG. 3B, top and middle panels). As a result,
the pooled population of nucleic acid tagged compounds is split
into a plurality of X.sub.2 sub-populations of distinct nucleic
acid tags. The number of sub-populations in the second step
(X.sub.2) may he the same or different than the number of
sub-populations resulting from the first stage split (X.sub.1). As
above, each sub-population of nucleic acid tagged molecules is
determined by the "second-position" hybridization sequence of the
nucleic acid tags (e.g., a.sub.2, b.sub.2, or c.sub.2) (FIG. 3B,
middle panel).
[0136] Each of the different "second-position" sub-populations of
nucleic acid tagged compounds is then reacted with one of a second
plurality of chemical monomers, a different chemical monomer for
each subset (e.g., A.sub.2, B.sub.2, or C.sub.2) (FIG. 3B, middle
panel). The result is a X.sub.2 different sub-populations of
nucleic acid tags, each population having a different chemical
monomer conjugated to the previous chemical monomer of each nucleic
acid tag-containing sub-population of molecules (FIG. 3B, bottom
panel). For example, as exemplified in the bottom panel of FIG. 3B,
nine different sub-populations of nucleic acid tag-containing
compounds can be generated, where a first population comprises the
chemical monomers A.sub.1 and A.sub.2, a second population
comprises the chemical monomers A.sub.1 and B.sub.2, a third
population comprises the chemical monomers A.sub.1 and C.sub.2, a
fourth population comprises the chemical monomers B.sub.1 and
A.sub.2, a fifth population comprises the chemical monomers B.sub.1
and B.sub.2, a sixth population comprises the chemical monomers
B.sub.1 and C.sub.2, a seventh population comprises the chemical
monomers C.sub.1 and A.sub.2, an eighth population comprises the
chemical monomers C.sub.1 and B.sub.2, and a ninth population
comprises the chemical monomers C.sub.1 and C.sub.2.
[0137] This process of splitting the previously reacted nucleic
acid tags into X.sub.n different sub-population (where X represents
the number of different capture sequences used to separate the
pooled compounds and n represents the step number of the synthetic
scheme) can be repeated as desired. For example, as illustrated in
FIGS. 3C and 3D, the nucleic acid tag-containing compounds can be
hybridized with a new set of immobilized capture oligonucleotides,
then reacting the X.sub.n separated sub-populations of tags with
X.sub.n different selected chemical monomers. These steps can be
repeated until all of the desired reaction steps are performed
successively on the reaction sites of the nucleic acid
tag-containing compound are complete (FIG. 3C and FIG. 3D). The
result is a combinatorial library of X.sub.1.times.X.sub.2.times. .
. . .times.X.sub.N different nucleic acid tagged chemical
compounds, wherein the particular of hybridization sequences at the
N positions (e.g., V.sub.1, V.sub.2, and V.sub.3, see FIG. 1) of
the nucleic acid tag of each compound dictates the sequence of
chemical monomers of the particular compound.
[0138] As exemplified in the top panel of FIG. 3D, twenty-seven
different populations of nucleic acid tagged compounds can be
generated from die steps as exemplified in FIGS. 3A-3C. The
exemplary combinatorial library of compounds includes, for example,
a first population comprising the chemical monomers A.sub.1,
A.sub.2, and A.sub.3, a second population comprising the chemical
monomers A.sub.1, B.sub.2, and A.sub.3, a third population
comprising the chemical monomers A.sub.1, C.sub.2, and A.sub.3, a
fourth population comprising the chemical monomers B.sub.1,
A.sub.2, and A.sub.3, a fifth population comprising the chemical
monomers B.sub.1, B.sub.2, and A.sub.3, a sixth population
comprising the chemical monomers B.sub.1, C.sub.2, and A.sub.3, a
seventh population comprising the chemical monomers C.sub.1,
A.sub.2, and A.sub.3, an eighth population comprising the chemical
monomers C.sub.1, B.sub.2, and A.sub.3, and a ninth population
comprising the chemical monomers C.sub.1, C.sub.2, and A.sub.3,
etc.
[0139] As exemplified in FIG. 1, the nucleic acid tag is composed
of Z.sub.n (e.g., n=9) regions of different catenated nucleic acid
sequences and a chemical reaction site. Five of these regions are
denoted C.sub.1 through C.sub.5 and refer to the "constant" or
"spacer" sequences that are the same for the nucleic acid tags. The
four remaining Z regions are denoted V.sub.1 through V.sub.4 and
refer to the "variable" hybridization sequences at the first
through fourth positions. In representative embodiments, the V
regions and C regions alternate in order from the 3' end of the
nucleic acid tag to the 5' end of the nucleic acid tag. In certain
embodiments, the first Z region is a C region. in other
embodiments, the first Z region is a V region. In certain
embodiments, the last Z region is a C region. In other embodiments,
the last Z region is a V region.
[0140] The variable hybridization sequences are generally different
for each group of sub-population of nucleic acid tags at each
position. In this embodiment, every V region is bordered by two
different C regions. As will be appreciated from below, all of the
V-region sequences are orthogonal, such that no two V-region
sequences cross-hybridize with each other. For example, in an
embodiment that comprises nucleic acid tags that include four
variable regions and 400 different nucleic acid sequences for each
of the four variable regions, there are a total of 1,600 orthogonal
nucleic acid hybridization sequences. Such hybridization sequences
can be designed according to known methods. For example, where each
variable hybridization sequence comprises 20 nucleotides, with a
possibility of one of four nucleotides at each position, 4.sup.20
different sequences are possible. Of the different possible
candidates, specific sequences can be elected such that each
sequence differs from another sequence by at least 2 to 3, or more,
different internal nucleotides.
[0141] In general suitable C and V regions comprise from about 10
nucleotides to about 30 nucleotides in length, or more. In certain
embodiments, C and V regions comprise from about 11 nucleotides to
about 29 nucleotides in length, including from about 12 to about
28, from about 13 to about 27, from about 14 to about 26, from
about 14 to about 25, from about 15 to about 24, from about 16 to
about 23, from about 17 to about 22, from about 18 to about 21,
from about 19 to about 20 nucleotides in length. In representative
embodiments C and V regions comprise about 20 nucleotides in
length.
[0142] A nucleic acid tag can comprise from about 1 to about 100 or
more different V regions (.hybridization sequences), including
about 200, about 300, about 500, or more different V regions. In
representative embodiments, a nucleic acid tag comprises from about
1 to about 50 different V regions, including about 2 to about 48,
about 3 to about 46, about 4 to about 44, about 5 to about 42,
about 6 to about 40, about 7 to about 38, about 8 to about 36,
about 9 to about 34, about 10 to about 32, about 11 to about 30,
about 12 to about 29, about 13 to about 28, about 13 to about 28,
about 14 to about 27, about 15 to about 26, about 16 to about 25,
about 17 to about 24, about 18 to about 23, about 19 to about 22.,
about 20 to about 21 different V regions.
[0143] A nucleic acid tag can comprise from about 1 to about 100 or
more different C regions (constant sequences), including about 200,
about 300, about 500, or more different C regions. In
representative embodiments, a nucleic acid tag comprises from about
1 to about 50 different C regions, including about 2 to about 48,
about 3 to about 46, about 4 to about 44, about 5 to about 42,
about 6 to about 40, about 7 to about 38, about 8 to about 36,
about 9 to about 34, about 10 to about 32, about 11 to about 30,
about 12 to about 29, about 13 to about 28, about 13 to about 28,
about 14 to about 27, about 15 to about 26, about 16 to about 25,
about 17 to about 24, about 18 to about 23, about 19 to about 22,
about 20 to about 21 different C regions.
[0144] The nucleic acid tags are synthesized such that regions
Z.sub.1 through Z.sub.n (e.g., n=9) are linked to each other
beginning with Z.sub.1 at the 3' and continuing in order with the
chemical reaction site at the 5' end following Z.sub.n. For
example, beginning with the 3' end of the nucleic acid tag, Z.sub.1
is linked to Z.sub.2, Z.sub.2 is linked to Z.sub.3, Z.sub.3 is
linked to Z.sub.4, etc., and chemical reaction site is linked to
Z.sub.n at any site on the nucleic acid tag, including the 3'
terminus, the 5' terminus, or any other position on the nucleic
acid tag.
[0145] As noted above, a population of nucleic acid tags is
degenerate, i.e., almost all of the nucleic acid tags differ from
one another in nucleotide sequence. The nucleotide differences
between different nucleic acid tags reside entirely in the
hybridization sequences (V regions). For example, an initial
population of nucleic acid tags can comprise of 400 first
sub-populations of nucleic acid tags based on the particular
sequence of V.sub.1 of each sub-population. As such, the V.sub.1
region of each sub-population comprises of any one of 400 different
20 base-pair hybridization sequences. Separation of such a
population of nucleic acid tags based on V.sub.1 would result in
400 different sub-populations of nucleic acid tags. Likewise, the
same initial population of nucleic acid tags can also comprise of
400 second subpopulations of nucleic acid tags based on the
particular sequence of V.sub.2 of each subpopulation, wherein the
second sub-populations are different than the first
subpopulations.
[0146] In the exemplary population of nucleic acid tags
demonstrated in FIG. 1, the first few of the first hybridization
sequences are denoted as a.sub.1, b.sub.1, c.sub.1 . . . j.sub.1,
in the V.sub.1 region of the different nucleic acid tags. Likewise,
the first few of the second hybridization sequences are denoted as
a.sub.2, b.sub.2, c.sub.2 . . . j.sub.2, in the V.sub.2 region of
the different nucleic acid tags. The first few of the third
hybridization sequences are denoted as a.sub.3, b.sub.3, c.sub.3 .
. . j.sub.3, in the V.sub.3, etc.
[0147] In certain embodiments, the nucleic acid tags share the same
twenty base-pair sequence for designated spacer regions while
having a different twenty base-pair sequence between different
spacer regions. For example, the nucleic acid tags comprise the
same C.sub.1 spacer region, the same C.sub.2 spacer region, and the
same C.sub.3 spacer region, wherein C.sub.1, C.sub.2, and C.sub.3
are different from one another.
[0148] Thus each 180 nucleotide long nucleic acid tag consists of
an ordered assembly of 9 different twenty base-pair regions
comprising the 4 variable regions (a.sub.1, b.sub.1, c.sub.1 . . .
d.sub.5, e.sub.5, f.sub.5, . . . h.sub.10, i.sub.10, j.sub.10) and
the 5 spacer regions (z.sub.1 . . . z.sub.11) in alternating order.
The twenty base-pair regions have the following properties: (i)
micromolar concentrations of all the region sequences hybridize to
their complementary DNA sequences efficiently in solution at a
specified temperature designated Tm, and (ii) the region sequences
are orthogonal to each other with respect to hybridization, meaning
that none of the region sequences cross-hybridizes efficiently with
another of the region sequences, or with the complement to any of
the other region sequences, at the temperature Tm.
[0149] The degenerate nucleic acid tags can be assembled from their
constituent building blocks by the primerless PCR assembly method
described by Stemmer et al., Gene (1995) 164(1) 49-53 or by
ligation strategies.
[0150] As noted above the nucleic acid tags further comprise a
chemical reaction site, including the 3' terminus, the 5' terminus,
or any other position on the nucleic acid tag. In some embodiments,
the chemical reaction site can be added by modifying the 5' alcohol
of the 5' base of the nucleic acid tag with a commercially
available reagent which introduces a phosphate group tethered to a
linear spacer, e.g., a 12-carbon chain terminated with a primary
amine group (e.g., as available from Glen Research, or numerous
other reagents which are available for introducing thiols or other
chemical reaction sites into synthetic DNA).
[0151] The chemical reaction site is the site at which the
particular compound is synthesized dictated by the order of V
region sequences of the nucleic acid tag. An exemplary chemical
reaction site is a primary amine. Many different types of chemical
reaction sites in addition to primary amines can be introduced at
any site, including the 3' terminus, the 5' terminus, or any other
position on the nucleic acid tag. Exemplary chemical reaction sites
include, but are not limited to, chemical components capable of
forming amide, ester, urea, urethane, carbon-carbonyl bonds,
carbon-nitrogen bonds, carbon-carbon single bonds, olefin bonds,
thioether bonds, and disulfide bonds. In the case of enzymatic
synthesis, co-factors may he supplied as are required for effective
catalysis. Such co-factors are known to those of skill in the art.
An exemplary cofactor is the phosphopantetheinyl group useful for
polyketide synthesis.
[0152] An entire compound library is synthesized by carrying out
alternate rounds of DNA-templated library splitting and chemical
andior biochemical coupling to each subsets of nucleic acid
tags.
[0153] The plurality of chemical compounds produced are linked to
nucleic acid sequence tags which facilitate identification of the
chemical structure. Conventional DNA sequencing methods are readily
available and useful for a determination of the sequence of the
synthesis-directing nucleic acid tags. (See, Maniatis et al., eds.,
"Molecular Cloning: A Laboratory Manual", Second Edition, Cold
Spring Harbor, N.Y. (1989)).
[0154] The compound library may be screened for a desired activity,
for example the ability to catalyze a particular reaction or to
hind with high affinity to an immobilized receptor. In most cases,
the subpopulation of molecules with the desired activity, as well
as their nucleic acid tags, are physically partitioned away from
siblings during the selection. Following selection, the nucleic
acid tags attached to the selected molecules are synthesized by the
polymerase chain reaction ("PCR") (Saiki et al., Science (1988)
239(4839) 487-491). The 5'hydroxyl of the 5'-end primer used to
synthesize the coding strand is modified with a phosphate group
tethered to a fresh primary amine chemical reaction site. After
synthesis, the coding strand is separated from the non-coding
strand. Because the nucleic acid tags direct the library synthesis,
rather than merely reporting on the synthetic history of individual
compounds, the coding strands amplified from the first library can
be used to direct the construction of a second generation compound
library. Iteration of this procedure, by carrying out multiple
rounds of selection. DNA tag amplification, and library
resynthesis, allows individual desirable compounds to be amplified
from extremely complex libraries.
[0155] An entire compound library or individual library members
produced by the above may be evaluated for one or more desired
activities in screening assays capable of distinguishing compounds
which modulate an activity or possess a desired structural or
functional property. Exemplary assays and functional analyses
include, but are not limited to, enzymatic assays, non-enzymatic
catalytic assays, protein-protein binding assays, receptor/ligand
binding assays and cell-based assays. More specifically, exemplary
cell-based methods are based on; (1) differential binding of
library compounds to a cell surface (i.e., binding to cancer cell
and not a non-cancer cell); (2) binding of library compounds to
components of a cell extract (e.g., binding to a cell fraction
produced by separating an entire cell extract on a sucrose
gradient); (3) library compounds capable of endocytosis by a cell
and (4) in vivo localization and binding properties of library
compounds by injecting the library into an animal. (See, e.g., Arap
et al., Science (1998) 279(5349) 377-80 which describes in vivo
selection of phage display libraries to isolate peptides that home
specifically to tumor blood vessels). As will be appreciated by
those of skill in the art, such assays may be performed on entire
libraries of compounds synthesized by the methods described herein
or sub populations derived therefrom.
[0156] The number of possible recognition compounds for which
ligands may be synthesized and identified by DPCC is virtually
unlimited. Recognition compounds include, but are not limited to,
oligonucleotides, single stranded RNA, single stranded DNA, DNA
binding proteins, RNA binding proteins, peptide nucleic acids,
peptides, depsipeptides, polypeptides, antibodies, peptoids,
polymers, polysiloxanes, inorganic compounds of molecular weight
greater that 50 daltons, organic compounds of molecular weight
between about 3000 daltons and about 50 daltons or combinations
thereof.
[0157] Desired ligands produced by the nucleic acid tag-directed
combinatorial library methods include, but are not limited to,
oligonucleotides, single stranded RNA, single stranded DNA, DNA
binding proteins, RNA binding proteins, peptide nucleic acids,
peptides, depsipeptides, polypeptides, antibodies, peptoids,
polymers, polysiloxanes, inorganic compounds of molecular weight
greater that 50 daltons, organic compounds of molecular weight
between about 3000 daltons and about 50 daltons or combinations
thereof.
[0158] In addition to allowing amplification of selected library
members, the method permits evolution of the encoded compound
libraries. More specifically, genetic recombination between the
nucleic acid tags which encode selected subpopulations of compounds
is carried out in vitro by mutagenesis or random fragmentation of
the nucleic acid tag sequence, followed by the generation of
related nucleic acid sequences ("gene shuffling", Stemmer, Nature,
(1994) 370 389-391; Stemmer et al., U.S. Pat. No. 5,811,238) and
subsequent step-wise synthesis of additional compounds. iteration
of this procedure, by carrying out multiple rounds of selection,
DNA tag amplification, genetic recombination and library
resynthesis, allows individual desirable compounds to evolve from
extremely complex libraries.
[0159] In some embodiments, a unique restriction site is introduced
into each specific hybridization sequence. By way of example,
partial digestion of a library with 11 specific hybridization
sequences is accomplished by partial digestion with 11
corresponding restriction enzymes, followed by a primerless PCR
reassembly reaction, allowing the nucleic acid tags for compounds
that have been selected out of the library to be recombined with
one another and further synthetic steps carried out. By analogy to
gene shuffling for protein synthesis (Crameri et al., Nature (1998)
391 288-291), the ability to carry out genetic recombination of
compound libraries vastly increases the efficiency with which the
diversity in the compound libraries can be explored and optimized.
In some embodiments, the gene has been circularized.
[0160] Accordingly, polynucleotide shuffling yields a population of
variant nucleic acid sequences, capable of directing the synthesis
of structurally-related, and/or functionally-related molecules,
and/or variants thereof to create ligands having one or more
desired activities. For example, molecules capable of binding to
the 5' untranslated region (UTR) of mRNA may be identified in this
manner. In vitro amplification of a selected. subpopulations of
synthesis directing nucleic acid tags by PCR, either prior to or
following "gene shuffling" is also possible using the methods
described above.
[0161] In still another aspect, a device is provided. The device
encompasses two arrays which include separate blocks. The block of
the first array encompasses two or more addressable wells which
include monoliths with attached recognition compounds which
selectively bind ligands. The block of the second array encompasses
two or more addressable wells which include ion exchange material.
The wells which include monoliths with attached recognition
compounds which selectively bind ligands are aligned with the wells
including the ion exchange material. In some embodiments, the ion
exchange material includes a monolith with ion exchange groups. In
other embodiments, the ion exchange material includes a monolith
with an anion exchange groups. Specific embodiments of any of the
arrays described above may be used in the device reported
herein.
[0162] As such, the above disclosure represents a novel device for
performing DPCC (see e.g., Harbury et al., U.S. Patent Application
No. US2006/0099626; Weisenger et al., PLoS ONE 7, e32299 for
previous examples). As described above, a DNA library is translated
into small molecules through repeated hybridization and chemistry
cycles. The number of cycles depends upon the number of
combinatorial chemistry steps that are required to make the
library. Each synthon at a particular step will correspond to a
unique codon. In the above embodiments, a hybridization array
separates (i.e., route) the DNA library such that members of a DNA
library (i.e., ligands) containing the same codon will be
immobilized on one or more monoliths, which are attached to the
cognate anti-codon recognition compound, into addressable wells.
The hybridization array may be used in place of the splitting
filters disclosed in Harbury et al., U.S. Patent Application No.
US2006/0099626 or the sepharose columns described in Weissenger et
al., PLoS ONE 7, e32299.
[0163] In another aspect, the hybridization array separates (i.e.,
route) the DNA library such that members of a DNA library (i.e.,
ligands) containing the same codon are immobilized on one or more
monoliths, which are attached to the cognate anti-codon recognition
compound, into addressable wells. A transfer array elutes the
specifically hybridized members of the DNA library into separate
addressable wells for further chemistry steps. Those of skill in
the art will appreciate that use of such a device is not restricted
to DNA libraries. Various diverse libraries of compounds can be
routed in the same fashion with appropriately chosen recognition
compounds and may be further processed as described below.
[0164] An exemplary hybridization array is illustrated in FIG. 4
and includes five plates assembled from top to bottom: A12, D11,
T1, D11 and A11. Plate T1 is an array where each addressable well
contains a monolith with an attached specific anticodon recognition
compound. Plates D11 and D12 are fabricated, for example, from a
plastic containing holes that connect the monoliths with attached
specific anticodon recognition compounds on plate T1 with the
grooves on the inner faces of plates A11 and A12. The grooves are
connected to channels through which air pressure and vacuum may be
applied. Diaphragms placed between the A and D plates create a
sealed continuous serpentine chamber consisting of the grooves on
the A plates, the holes in the D plates, and the monoliths in the T
plate. One of the D plates (D11) has ports through which liquid can
enter and leave this chamber. The grooves in the top and bottom A
plates are designed such that appropriate cyclical application of
air pressure and vacuum to the channels in the A plates sets up a
directional flow of the liquid in the serpentine chamber by, for
example, a hybridization pump (see e.g., Weissenger et al., PLoS
ONE 7, e32299). The net effect of the mesofluidic device may be
likened to flowing the library of DNA ligands through an array of
monolith columns attached to a specific anticodon recognition
compound (i.e., the hybridization array), which are connected in
series in a head-to-tail fashion. The skilled artisan will
appreciate that N such devices may be connected in series so that a
library can be partitioned, in principle, between an infinite
number of hybridization array.
[0165] As the DNA library flows through the hybridization array,
members of the DNA library containing the appropriate codon will
hybridize to a monolith attached to a cognate anticodon recognition
compound in the hybridization array. In some embodiments, cycling
the DNA library through the device five times will ensure >90%
partitioning of each codon (i.e., 90% of the DNA library with each
codon is immobilized on the correct monolith with attached
anticodon recognition compounds; the above requires that each pass
of the DNA library through the monolith columns with attached
anticodon recognition compounds partitions >40% of the cognate
codon). Accordingly, at the end of this step, in some embodiments,
>90% of the library will be immobilized on the hybridization
array. In other embodiments, cycling the DNA library through the
device five times will ensure between about 80% and about 90%
partitioning of each codon Accordingly, at the end of this step, in
some embodiments, between about 80% and about 90% of the library
will be immobilized on the hybridization array at the end of this
step. In some embodiments, the device is disassembled and the
hybridization array that comprises monolith attached to a cognate
anticodon recognition compound which have bound specific members of
the DNA library (i.e., plate T1) are now used to form a transfer
array.
[0166] The transfer array allow parallel processing of the
individual monolith columns in the array where the monoliths with
attached anticodon recognition compounds have hybridized to
specific members of the DNA library. In some embodiments, the
transfer array includes plates D01, T1 and D02, as illustrated in
FIG. 5. In some of these embodiments, silicone gaskets, are placed
in the street pattern grooves on the inner face of plates D01 and
D02 (face adjacent to T1) to prevent cross contamination of the
addressable transfer array wells. Liquid applied to top of the D01
plate will be drawn through the assembly, for example, by
centrifugation. The transfer array allows, washing, elution, and
regeneration of the array with monoliths with attached anticodon
recognition compounds. In some embodiments, the wells of the
transfer array will contain ion exchange material which may be used
to immobilize the specific members of the DNA library, which were
hybridized to the monoliths with attached anticodon recognition
compounds. In some embodiments, the address of the transfer array
wells corresponds directly to the address of the hybridization
array wells.
[0167] In addition, while the Figures illustrate the first routed
codon towards the 3' end, the skilled artisan will appreciate that
the first routed codon can also be routed to the 5' end. Those of
skill in the art will also appreciate that more than one chemical
step can follow each routing step.
[0168] Finally, it should be noted that there are alternative ways
of implementing the present invention. Accordingly, the present
embodiments are to be considered as illustrative and not
restrictive, and the invention is not to be limited to the details
given herein, but may be modified within the scope and equivalents
of the appended claims.
[0169] All publications and patents cited herein are incorporated
by reference in their entirety to disclose and describe the methods
and/or materials in connection with which the publications are
cited. It is understood that the present disclosure supersedes any
disclosure of an incorporated publication to the extent there is a
contradiction. The publications discussed herein are provided
solely for their disclosure prior to the filing date of the present
application. Nothing herein is to he construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0170] The following examples are provided for illustrative
purposes only and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
Titanium Silanization
[0171] Two #4 titanium grade 5 washers (United Titanium, outer
diameter=0.3125 inch, inner diameter=0.125 inch, thickness=0.032
in) were threaded with a #6-32 tap (major diameter =0.136 inch, 32
threads/inch), washed with water, washed with acetone and then
dried for 5 minutes. Each washer was then placed in an eppendorf
tube and about 250 .mu.l of a solution of 115 .mu.l MPS
([3-(Methacryloyloxy)propyl]trimethoxysilane from Gelest) and 40
.mu.l titanium n-butoxide (Gelest) dissolved in 345 .mu.l heptane
was then added, the tube shaken and turned upside down. After about
15 minutes, the washers were air-dried for 15-30 minutes, cured at
99.degree. C. for 45 minutes, rinsed with acetone to remove excess
unreacted reagents and air-dried for an additional 5 minutes.
Example 2
Monolith Formation and Bonding to Silanized Titanium
[0172] The silanized titanium washers were placed in a foil bag
(Sigma #183385), the bag was flushed 5 times with Na and then
closed. A mixture including 3 mL GMA, 1 mL EDMA, 3 mL 1-propanol,
2.4 mL 1,4-butanediol and 0.6 mL of water was sparged with N.sub.2
for about 2 minutes, sonicated for about 10 minutes, at which time
10 mgs of AIBN (2,2'-azobis(2-methylpropionitrile) was added and
the mixture was swirled to dissolve the initiator. The mixture was
sonicated for an additional 1 minute, the head-space was purged
with N.sub.2 for about 30 sec, poured into the foil bag containing
the silanized washers and sealed after removing any residual
air/N.sub.2. The washers were sandwiched between two outer aluminum
plates, which ensured that the polymer only formed within the
threaded holes ("wells") in the washers. The assembly was placed
horizontally in the oven at 60.degree. C. and incubated overnight.
The plate/clamp assembly was removed from the oven, allowed to
equilibrate to room temperature and dismantled to provide the
monolith washers. Excess monolith was removed from the edges of the
washer.
[0173] The monolith washer was placed in a filter-housing attached
to a syringe and washed with 5 mL ethanol and 5 mL water.
Example 3
Silanization of Aluminum 6061 Array
[0174] A 3.125 mm thick aluminum plate of 6061 grade which had been
machined to form a 384 well plate was washed with tap water, rinsed
in distilled water for 5 minutes, washed in acetone for 15 minutes
and air dried for 5 minutes. The plate was treated with 10% NaOH
for about 10 minutes at about 45.degree. C., blotted to remove
excess NaOH and immersed in a 30% HNO.sub.3 solution, until the
entire surface of the plate was silver (1-3 minutes). The plate was
then rinsed with distilled water, air dried for between about 10-60
minutes and treated with 30% MPS in acetone at 60.degree. C. for 15
minutes, washed three times in acetone, air-dried and cured at
80.degree. C. overnight. The plate was then washed with acetone and
air-dried for about 10-15 minutes.
Example 4
Monolith Formation and Bonding to Silanized Aluminum 6061 Array
[0175] The silanized aluminum plate prepared in Example 3 was
placed in a foil bag (Sigma #Z183393), the bag was flushed 5 times
with N.sub.2 and then closed. A mixture including 15 mL GMA, 5 mL
EDMA, 15 mL 1-propanol, 12 mL 1,4-butanediol and 3 mL of water was
sparged with N2 for about 5 minutes, sonicated for about 10
minutes, at which time 25-50 mgs of AIBN was added and the mixture
was swirled to dissolve the initiator. The mixture was sonicated
for an additional 1 minute, the head-space was purged with N2 for
about 1 minute, poured into the foil bag containing the silanized
aluminum plate and sealed after removing any residual air/N.sub.2.
The enclosed silanized aluminum plate was then sandwiched between
two outer aluminum blocks which were secured with C-clamps. This
ensured that the polymer only formed within the machined holes
("wells") in the plate. The assembly was placed horizontally in the
oven at 60.degree. C. and incubated overnight. The plate/clamp
assembly was removed from the oven, allowed to equilibrate to room
temperature and dismantled to provide the monolith array plate.
Excess monolith was removed from the edges of the plate.
[0176] The monolith array was washed three times with between about
30 mL and about 40 mL of ethanol using vacuum (20 psi for between
10-60 seconds) to draw the ethanol through the monoliths attached
to the aluminum plate. Then, between about 30 .mu.l and about 40
.mu.l of ethanol are added to each well and drawn through the
monolith by application of vacuum (15-20 psi for between about 15
and about 20 seconds). Finally, between about 30 ul and about 40
.mu.l of ethanol are added to each well and drawn through the
monolith by a 1000 rpm spin for about 3 minutes. The monolith array
was then washed with water in a similar manner Ideally, every
monolith in every well is permeable to solvent. A single
impermeable well is enough to disqualify the array from further
use.
Example 5
Azide Opening of Glycidyl Epdxide Monolity Attached to Aluminum
6061 Array
[0177] Briefly, 20 .mu.l of an aqueous solution of sodium azide (25
mmol in 8 mL of water and 4.6 mL of glacial acetic acid) was added
to each well of the monolith array prepared in Example 4 and drawn
through the monolith using vacuum. The above procedure was repeated
once, the array was transferred to a bath containing the above
azide solution, degassed for about five minutes, transferred to a
reservoir filled with the above azide solution, incubated at
30.degree. C. overnight, washed extensively with distilled water
and stored in water to provide an azido alcohol functionalized
monolith array.
Example 6
Attachment of an Oligonucleotide to Azido Functionalized Monolith
Attached to Aluminum 6061 Array
[0178] 30 ul of `click` solution containing 0.1 M sodium phosphate,
pH 7.0, 625 uM CuSO4, 3.125 mM THPTA
(Tris(3-hydroxypropyltriazolylmethyl)amine, Sigma-Aldrich), 12.5 mM
aminoguanidine-HCl, 0.3M NaCl, 12.5 mM ascorbate, and 1 nmol of the
oligonucleotide to be immobilized (general structure
5'hexynyl-TTTTTTTTTT-anticodon, purchased from Eurofins MWG Operon
Inc., 2211 Seminole Drive Huntsville, Ala. 35805) was added to each
azido-alcohol functionalized monolith in the array and incubated
for a total of 1 hour. The `click` solution was periodically
(approximately, every 15 minutes) centrifuged out of the monolith,
replenished with fresh ascorbate (addition of up to 3 ul of 100 mM
ascorbate), and added back to the same monolith. After 1 h
incubation, the `click` solution was centrifuged out of the
monolith, and the array was washed three times with 30 .mu.l of
Tris-EDTA buffer (10mM Tris-HCl, pH8.0, 1 mM EDTA) to chelate
copper and quench the reaction. The resultant functionalized arrays
were stored in 1 mM EDTA, 0.02% azide at 4.degree. C.
[0179] The efficiency of click chemistry in attaching the
5'-hexynyl-oligo to the azide-modified monolith was assessed by
removal of the oligo from the `click` solution. Typically, the test
reaction, consisting of equal concentrations of two oligos, a 5'-OH
oligo (reference oligo) and the 5'-hexynyl oligo (test oligo), was
incubated with the monolith in the `click buffer` for a total of 1
hour at room temperature, after which it was centrifuged out of the
monolith. The exhausted `click` solution was analyzed by RP-HPLC on
a C18 column with an acetonitrile gradient (mobile phase A: 135 mM
triethylamine, 150 mM glacial acetic acid, 5% acetonitrile; mobile
phase B: 25% mobile phase A, 75% acetonitrile) which separated the
two oligos. The relative peak areas of the reference and the test
oligos in the samples before and after incubation with the monolith
were used to estimate the proportion of the 5'hexynyl-oligo that
was `clicked` onto the monolith.
Example 7
Propiolic Acid Blocking of Oligonucleotide Functionalized Monolith
Attached to Aluminum 6061 Array
[0180] Any unreacted azide groups in the monolith array treated as
in example 6 were blocked by reacting with propiolic acid as
follows. 30 ul of `click` solution contained 0.1M sodium phosphate,
pH 7.0, 625 uM CuSO4, 3.125 mM THPTA
(Tris(3-hydroxypropyltriazolylmethyl)amine, Sigma-Aldrich), 12.5 mM
aminoguanidine-HCl, 0.3 M NaCl, 12.5 mM ascorbate, and 8 mM
propiolic acid was added to each functionalized monolith in the
array and incubated for a total of 30 minutes. The `click` solution
was periodically (approximately every 15 min) centrifuged out of
the monolith, replenished with fresh ascorbate (addition of up to 3
ul of 100 mM ascorbate), and added back to the same monolith. After
30 minutes incubation, the `click` solution was centrifuged out of
the monolith, and the array was washed three times with 30 ul of
Tris-EDTA buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA) to chelate
copper and quench the reaction. The treated arrays were stored in 1
mM EDTA, 0.02% azide at 4.degree. C.
Example 8
Silanization of Aluminum 5038 Array
[0181] Silanization was performed as in Example 3, except the plate
was treated with about 10% NaOH for about 15 minutes at about
45.degree. C.
Example 9
Monolith Formation and Bonding to Silanized Aluminum 5038 Array
[0182] The procedure of Example 4 was used to provide the above
monolith array.
Example 10
UV Treatment of Peek Washers
[0183] The washers were threaded using #10-32 tap (major
diameter=0.19 inch, threads/inch=32) and submerged in a 1:10
EDMA:methanol solution in a scintillation vial. Each washer had a
stainless steel cone at its center to disperse incident UV light.
Also, cones were placed around the side of the washer to use the
outer diameter face as a test for bonding strength. The vials were
then immersed in the EDMA:methanol solution to a height of about 1
cm. The washers were then irradiated with 302 nm UV light for 15
minutes from a distance of about 3 cm, flipped and irradiated again
with 302 nm UV light for 15 minutes from a distance of about 3 cm,
rinsed 3.times. with methanol, air dried and stored in a foil bag
under nitrogen. The washers were used to cast monoliths according
to the procedure described in Example 2.
* * * * *
References