U.S. patent application number 10/915153 was filed with the patent office on 2008-12-25 for color-encoding and in-situ interrogation of matrix-coupled chemical compounds.
Invention is credited to Richard H. Ebright, Michael Seul.
Application Number | 20080318211 10/915153 |
Document ID | / |
Family ID | 21949186 |
Filed Date | 2008-12-25 |
United States Patent
Application |
20080318211 |
Kind Code |
A9 |
Seul; Michael ; et
al. |
December 25, 2008 |
Color-encoding and in-situ interrogation of matrix-coupled chemical
compounds
Abstract
Disclosed is a method for the physico-chemical encoding of a
collection of beaded resin ("beads") allowing determination of the
chemical identity of bead-anchored compounds, following
identification of beads bearing compounds of interest in an assay,
by in-situ interrogation of individual beads, which does not
require isolation of the beads of interest. These methods can be
used to implement color-coding strategies in applications and
including the ultrahigh-throughput screening of bead-based
combinatorial compounds libraries as well as multiplexed diagnostic
and environmental testing and other biochemical assays.
Inventors: |
Seul; Michael; (Fanwood,
NJ) ; Ebright; Richard H.; (North Brunswick,
NJ) |
Correspondence
Address: |
ERIC P. MIRABEL
35 TECHNOLOGY DRIVE
SUITE 100
WARREN
NJ
07059
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20050059063 A1 |
March 17, 2005 |
|
|
Family ID: |
21949186 |
Appl. No.: |
10/915153 |
Filed: |
August 9, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09448420 |
Nov 22, 1999 |
7083914 |
|
|
10915153 |
Aug 9, 2004 |
|
|
|
PCT/US98/10719 |
May 22, 1998 |
|
|
|
09448420 |
Nov 22, 1999 |
|
|
|
60047472 |
May 23, 1997 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/6.13; 435/6.14; 435/7.1 |
Current CPC
Class: |
C40B 20/04 20130101;
B01J 2219/00545 20130101; C40B 40/10 20130101; G01N 21/29 20130101;
G01N 21/6458 20130101; G01N 33/54313 20130101; C40B 50/04 20130101;
B01J 2219/00659 20130101; B01J 2219/0059 20130101; C40B 50/16
20130101; B01J 2219/00648 20130101; G01N 33/582 20130101; B01J
2219/00596 20130101; C40B 30/04 20130101; B01J 2219/00459 20130101;
G01N 33/6845 20130101; C40B 40/06 20130101; B01J 2219/00707
20130101; G01N 2021/1765 20130101; B01J 2219/00725 20130101; B01J
2219/005 20130101; C40B 70/00 20130101; B01J 2219/00722 20130101;
B01J 2219/00592 20130101; G01N 21/64 20130101; B01J 19/0046
20130101 |
Class at
Publication: |
435/006 ;
435/007.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53; G01N 33/567 20060101
G01N033/567 |
Claims
1. A method of identifying a compound having a selected property of
interest in a library of compounds, each of said compounds being
bound to its respective solid support, and being produced by a
unique reaction series composed of N reaction steps, wherein each
compound is prepared from a component, and N is an integer from at
least 1 to about 100, which comprises: a) dividing a population of
solid supports having at least one type of a first functional group
at the surface of said solid support selected from the group
consisting of CO.sub.2H, OH, SH, NH2, NHR, CH.sub.2Cl, CH.sub.2Br
and CHN.sub.2, wherein R is a linear C.sub.1-C.sub.9 alkyl group,
into M batches, wherein M is an integer from at least 2 to about
25; b) coupling the M batches of solid support in a set of at least
one reaction respectively with M different components so as to form
a bond with the solid support via said first functional group, said
components being independently protected or unprotected; c) adding
to each batch, either prior to coupling step b), concurrently
therewith, or subsequently to step b), from about 0.001 to about
0.5 molar equivalent of a spectrally distinguishable fluorophore
tag associated uniquely with each component, said tag being
identified by its characteristic excitation wavelength(s), emission
wavelength(s), excited state lifetime and emission intensity, said
tag being activated so as to be capable of forming either a direct
bond to the surface of the solid support, either via the first or a
second functional group which is protected or unprotected and is
the same as or different from the first functional group bonded to
the component, or an indirect bond via a C.sub.1-C.sub.9 linear or
branched alkyl linker moiety which is either interrupted or
uninterrupted by at least one oxygen or nitrogen atom or a
carbonyl, (C--O)NH or NH(C--O) moiety, wherein when said second
finctional group is protected, said fuictional group is deprotected
prior to forming said direct or indirect bond, said linker being
bonded to the second finctional group at the surface of the solid
support; and either d) recombining all M batches, said recombining
step being either prior to or subsequent to step e) and steps
e)-g); or e) performing an assay capable of indicating that any
compound in the library either while bound to or cleaved from its
solid support has the property of interest; f) collecting spectral
fluorescence data for each respective solid support so as to
determine respective relative abundances of the fluorophore tags
bound thereto; and g) analyzing the collected spectral fluorescence
data by comparing the respective relative abundances of the
fluorophore tags determined in step f) so as to determine the
unique reaction series for the compound, thereby identifying the
compound having the property of interest.
2. The method of claim 1 wherein the components are independently
selected from the group consisting of an amino acid, a hydroxyacid,
an oligoamino acid, an oligopeptide, a saccharide, an
oligosaccharide, a diamine, a dicarboxylic acid, an
amine-substituted sulfhydryl, a sulfhydryl-substituted carboxylic
acid, an alicyclic, an aliphatic, a heteroaliphatic, an aromatic
and a heterocyclic moiety.
3. The method of claim 2 wherein the saccharide is a suitably
protected D- or L- glucose, fructose, inositol, mannose, ribose,
deoxyribose or fucose.
4. The method of claim 2 wherein the oligopeptide is an enkephalin,
a vasopressin, an oxytocin, an atrial natrietic factor, a bombesin,
a calcitonin, a parathyroid hormone, a neuropeptide Y or an
endorphin, or a fragrnent thereof comprising at least 20% of the
components thereof, or an isosteric analogue thereof wherein
independently NH(C--O) is replaced by NH(C--O)NH,
NH(C--O)O,CH.sub.2(C--O) or CH.sub.2O; NH2 is replaced by OH, SH,
N0.sub.2 or CH.sub.3; CH.sub.3S is replaced by CH.sub.3 (S--O) or
CH.sub.3 CH.sub.2; indole is replaced by naphthyl or indene;
hydroxyphenyl is replaced to tolyl, mercaptophenyl or nitrophenyl;
and/or hydrogen in an aromatic ring is replaced by chlorine,
bromine, iodine or fluorine; C.sub.1-C.sub.4 alkyl is replaced by
partially or fully flourinated C.sub.1-C.sub.4 alkyl.
5. The method of claim 2 wherein the oligopeptide is an ACE
inhibitor, an HIV protease inhibitor, a cytolytic oligopeptide or
an antibacterial oligopeptide.
6. The method of claim 2 wherein the aromatic is para-disubstituted
benzene, biphenyl, naphthalene or anthracene, either substituted or
unsubstituted by linear or branched chain lower alkyl, alkoxy,
halogen, hydroxy, cyano or nitro.
7. The method of claim 2 wherein the heterocyclic moiety is
2,6-disubstituted pyridine, thiophene, 3-7-disubstituted
N-protected indole or 2,4-disubstituted imidazole, either
substituted or unsubstituted by linear or branched chain lower
alkyl, alkyl, halogen, hydroxy, cyano or nitro.
8. The method of claim 1 wherein the solid support is a
microsphere, a bead, a resin or a particle, and is composed of a
material selected from the group consisting of polystyrene,
polyethylene, cellulose, polyacrylate, polyacrylamide, or
preferably a silica to glass bead.
9. The method of claim 1 wherein the solid support is chemically
modified by covalent attachment of either a substituted or
unsubstituted oligo- or polyethyleneglycol, which either terminated
or unterminated by an amine substituted by either hydroxymethyl,
chloromethyl, aminomethyl or mercaptomethyl, wherein the functional
group at the surface of the solid support is hydroxy, chlorine, NH2
or SH, respectively.
10. The method of claim 1 wherein the assay is performed while the
compound is cleaved from its solid support under conditions whereby
the compound remains adsorbed to the solid support.
11. The method of claim 1 wherein the property of interest is
binding affinity of a compound to a receptor, the assay is
performed by determining a physical response to binding by a) first
admixing with the library of compounds a solution of a labelled
receptor so as to result in labelled receptor bound to at least one
compound bound to a solid support; b) removing the solution from
the solid support; and either c) washing the solid support so as
substantially to remove non-bound labelled receptor, and step (d),
or d) measuring the physical response due to bound labelled
receptor so as to determine the binding affinity.
12. The method of claim 11 wherein the receptor is labelled by a
fluorescent dye, a colored dye, radioisotope or an enzyme.
13. The method of claim 11 wherein the physical response is
fluorescence emission, optical absorption or radioactivity.
14. The method of claim 1 wherein the components have a structure
independently selected from the group consisting of: ##STR1##
wherein R, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R6 are
independently methyl, ethyl, linear or branched chain
C.sub.3-C.sub.9, phenyl, benzyl, benzoyl, cyano, nitro, halo,
formyl, acetyl and linear or branched chain C.sub.3-C.sub.9 acyl;
wherein a, b, c, d and e are independently 0, 1, 2 or 3; wherein X,
Y and Z are independently NH, 0, S, S(--O), CO, (CO)O, O(CO),
NH(C--O) or (C--O)NH; and wherein W is independently N, 0 or S.
15. The method of claim 1 wherein at least one component is an
amino acid, bearing a protected or unprotected group which is
capable of participating in a further reaction or coupling step and
is nitrogen, said protecting group being selected from the group
consisting of N-a-fluorenylmethyloxcarbonyl, t-butyloxycarbonyl,
t-amyloxycarbonyl, (trialkyisilyl) ethyloxycarbonyl, t-butyl and
benzyl.
16. The method of claim 1 wherein the fluorophore tag represents a
bit of a binary code, and comprises zero, one or more than one
fluorescent dye, multiple fluorescent dyes, said dye(s) being
spectrally distinguishable by excitation wavelength, emission
wavelength, excited-stated lifetime or emission intensity.
17. The method of claim 16 wherein emission intensity is
distinguished by adjusting the ration of the relative quantities of
each fluorophore.
18. The method of claim 17 wherein the ratio is 1:1, 2:1; 3:1 or
4:1.
19. The method of claim 1 wherein the fluorophore tags are dyes
selected from the group consisting of compounds with the chemical
names: 3-(c-carboxypentyl)-3'-ethyl-oxacarbocyanine-6,6'-disulfonic
acid 1 -(c-carboxypentyl)-1
'-ethyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'- disulfonic acid
1-(e-carboxypentyl)-1 '-ethyl-3,3,3',3'-tetramethyl-3H-
benz(e)indocarbocyanine-5,5',7,7'-tetrasulfonic acid 1
-(E-carboxypentyl)-1 '-ethyl-3,3
,3',3'-tetaamethylindocarbocyanine-5,5'- disulfonic acid
1-(E-carboxypentyl)-l'-ethyl-3,3,3',3'-tetramethyl-3H-
benz(e)indodicarbocyanine-5,5',7,7'-tetrasulfonic acid
1-(c-carboxypentyl)-1
'-ethyl-3,3,3',3'-tetramethylindotricarbocyanine-5,5'- disulfonic
acid and are activated as active esters selected from the group
consisting of succinimidyl, sulfosuccinimidyl, p-nitrophenol,
pentafluorophenol, HOBt and N-hydroxypiperidyl.
20. The method of claim 1 wherein the fluorophore tags are dyes
selected from the group consisting of compounds with the chemical
names:
6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)a-
mino) hexanoic acid
6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)
amino) hexanoic acid, 6-((4,4-difluoro-1
,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,
4a-diaza-s-indacene-2-propionyl) amino)hexanoic acid,
6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)
phenoxy) acetyl) amino)hexanoic acid,
6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)
styryloxy)acetyl) aminohexanoic acid, and
6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)
styryloxy) acetyl)aminohexanoic acid, and are activated as active
esters selected from the group consisting of succinimidyl,
sulfosuccinimidyl, p-nitrophenol, pentafluorophenol, HOBt and
N-hydroxypiperidyl.
21. The method of claim 1 wherein the fluorophore tags are dyes
selected from the group consisting of compounds with the chemical
structures: ##STR2## and are activated as active esters selected
from the group consisting of succinimidyl, sulfosuccinimidyl,
p-nitrophenol, pentafluorphenol, HOBt and N- hydroxypiperidyl.
22. The method of claim 1 wherein the assay is performed by
cleaving compounds from the solid support while permitting
diff-usion through solution and binding to receptors, said
receptors being arranged in proximity to each solid support.
23. The method of claim 1 wherein the fluorescence data are
collected from multiple solid supports using multi-spectral imaging
methods.
24. The method of claim 1 wherein one of the fluorophore tags
uniquely associated with a preselected component or reaction
comprises a ligand and a substance capable of binding specifically
to the ligand, said ligand being labelled with a fluorophore and
attached in a post-assay reaction, said substance being present on
the solid support and attached prior to, concurrently with, or
subsequent to the coupling of the component, whereby the labelled
ligand when bound to the substance indicates the presence of the
preselected component.
25. The method of claim 1 wherein the solid support is a polymeric
bead, and spectral fluorescence data is collected by a) forming
either a static planar array or a dynamic planar array of beads;
and b) obtaining a fluorescence image for each bead.
26. The method of claim 25 wherein the planar array of beads is
formed adjacent to the planar walls of a sandwich flow cell and
controlled by light-controlled electrokinetic means.
27. The method of claim 25 wherein the planar array of beads is
formed by using an apparatus capable of dynamically assembling and
dissembling an array of beads at an interface between an electrode
and an electrolyte solution, said apparatus comprising: i) an
electrode, an electrolyte solution and an interface therebetween
ii) a plurality of beads located in said electrolyte solutions;
iii) said electrode being patterned to include at least one area of
modified electrochemical properties; iv) an illumination source
which illuminates said electrode with a predetermined light
pattern; v) an electric field generator which generates an electric
field at said interface to cause the assembly of an array of beads
in accordance with the predetermined light pattern and the
electrochemical properties of said electrode; and vi) an electric
field removal unit which removes said electric field to cause the
dissembly of said array of beads.
28. The method of claim 25 wherein spectral fluorescence data are
collected for the bead array by initially forming a spatially
encoded array of beads suspended at an interface between an
electrode and an electrolyte solution, comprising the following
steps: i) providing an electrode and an electrolyte solution; ii)
providing multiple types of particles, each type being stored in
accordance with chemically or physically distinguishable particle
characteristics in one of a plurality of reservoirs, each reservoir
containing a plurality of like-type particles suspended in said
electrolyte solution; iii) providing said reservoirs in the form of
an mxn grid arrangement; iv) patterning said electrode to define
mxn compartments corresponding to said rnxn grid of reservoirs; v)
depositing mxn droplets from said mxn reservoirs onto said
corresponding mnn compartments, each said droplet originating from
one of said reservoirs and remaining confined to one of said nzxn
compartments and each said droplet containing at least one
particle; vi) positioning a top electrode above said droplets so as
to simultaneously contact each said droplet; vii) generating an
electric field between said top electrode and said man droplets;
viii) using said electric field to form a bead array in each of
said MxN compartments, each said bead array remaining spatially
confined to one of said mxn droplets; ix) illuminating said mxn
compartments on said patterned electrode with a predetermined light
pattern to maintain the position of said bead arrays in accordance
with said predetermined light and the pattern of mxn compartments;
and x) positioning said top electrode closer to said electrode
thereby fusing said mxn droplets into a continuous liquid phase,
while maintaining each of said mxn bead arrays in one of the
corresponding men compartments.
29. The method of claim 28, wherein said compartments are
hydrophilic and the remainder of said electrode surface is
hydrophobic.
30. The method of claim 1 wherein N is an integer from at least
2.
31. The method of claim 1 wherein N is an integer from at least 4
to about 12.
32. The method of claim 1 wherein M is an integer from at least 4
to about 10
33. The method of claim 1 wherein from about 0.01 to about 0.05
molar equivalent of a spectrally distinguishable fluorophore tag is
added in step c).
34. A compound having a selected property of interest as identified
in accord with claim 1.
35. A chemical library prepared in accord with claim 1.
36. An apparatus for identifying a compound having a selected
property of interest in a library of compounds, each of said
compounds being bound to its respective solid support, and being
produced by a unique reaction series composed of N reaction steps,
wherein each said compound is prepared from a component, and N is
an integer from at least 1 to about 100, said solid support being
at least one particle array, said apparatus comprising: a) an
electrode and an electrolyte solution having an interface
therebetween, b) an electric field generator which generates an
electric field at an interface between an electrode and an
electrolyte solution; c) said electrode being patterned to modify
the electrochemical properties of said electrode; d) an
illuminating source which illuminates said interface with a
predetermined light pattern to control the movement of said
particles in accordance with said predetermined light pattern and
the electrochemical properties of said electrode; e) means for
preparing said chemical library, which comprises: i) means for
dividing a population of solid supports having at least one type of
a first finctional group at the surface of said solid support
selected from the group consisting of CO.sub.2H, OH, SH, NH2, NHR,
CH.sub.2Cl, CH.sub.2Br and CHN.sub.2, wherein R is a linear
C.sub.1-Cg alkyl group, into M batches, wherein M is an integer
from at least 2 to about 25; ii) means for coupling the M batches
of solid support in a set of at least one reaction respectively
with Mdifferent components so as to form a bond with the solid
support via said first functional group, said components being
independently protected or unprotected; iii) means for adding to
each batch either prior to coupling step ii), concurrently
therewith, or subsequently to step ii), from about 0.001 to about
0.5 molar equivalent of a spectrally distinguishable fluorophore
tag associated uniquely with each component, said tag being
identified by its characteristic excitation wavelength(s), emission
wavelength(s), excited state lifetime and emission intensity, said
tag being activated so as to be capable of forming either a direct
bond to the surface of the solid support, either via the first or a
second functional group which is protected or unprotected and is
the same as or different from said first fuictional group, a direct
bond to the component which if protected is priorly deprotected, or
an indirect bond via a C.sub.1,-C.sub.9, linear or branched alkyl
linker moiety which is either interrupted or uninterrupted by at
least one oxygen or nitrogen atom or a carbonyl, (C--O)NH or
NH(C--O) moiety, wherein when said second functional group is
protected, said second functional group is deprotected prior to
forming said direct or indirect bond, said linker being bonded to
said second finctional group at the surface of the solid support;
and either iv) means for recombining all M batches, said
recombining step either being prior to or subsequent to step v),
and steps v)-vii); or; v) means for performing an assay capable of
indicating that any compound-in the library either while bound to
or cleaved from its solid support has the property of interest; vi)
means for collecting spectral fluorescence data for each respective
solid support so as to determine respective relative abundances of
the fluorophore tags bound thereto; vii) means for analyzing the
collected spectral fluorescence data by comparing the respective
relative abundances of the fluorophore tags determined in step vi)
so as to determine the unique reaction series for the compound,
thereby identifying the compound having the property of
interest.
37. A method of identifying a compound having a selected property
of interest in a library of compounds, each of said compounds being
bound to its respective solid support, and being produced by a
unique reaction series composed of N coupling or reaction steps,
wherein each compound is prepared from components which are
independently the same or different, and N is an integer from at
least 1 to about 100, which comprises: a) dividing a population of
solid supports having at least one type of a first functional group
at the surface of said solid support surface selected from the
group consisting of CO.sub.2H, OH, SH, NH2, NHR, CH.sub.2Cl, CH,Br
and CHN.sub.2, wherein R is a linear C.sub.1-C.sub.9 alkyl group,
into Mbatches, wherein M is an integer from at least 2 to about 50;
b) coupling the M batches of solid support in a set of at least one
reaction respectively with M different initial components so as to
form a bond with the solid support via said first functional group,
said components being protected or unprotected at a group which is
capable of participating in a further coupling step and
orthogonally protected at non-participating group(s); c) adding to
each batch either prior to coupling step b), concurrently
therewith, or subsequently to step b), from about 0.001 to about
0.5 molar equivalent of a spectrally distinguishable fluorophore
tag associated uniquely with each initial component or a reaction
of step b), said tag nbeing identified by its characteristic
excitation wavelength(s), emission wavelength(s), excited state
lifetime and emission intensity, said tag being activated so as to
be capable of forming either a direct bond to the surface of the
solid support, either via the first or a second functional group
which is protected or unprotected and is the same as or different
from said first functional group, a direct bond to the initial
component which if protected is priorly deprotected, or an indirect
bond via a C.sub.1-C.sub.9 linear or branched alkyl linker moiety
which is interrupted or uninterrupted by either at least one oxygen
or nitrogen atom or a carbonyl, (C--O)NH or NH(C--O) moiety, said
linker being bonded to said first functional group at the surface
of the solid support, wherein when said second functional group is
protected, said second functional group is deprotected prior to
forming said direct or indirect bond; and either d) recombining all
M batches and cleaving any protecting group present at a group
which is to participate in a further coupling step, said
recombining step being either prior to or subsequent to step e),
and steps e)-h); or e) iteratively N-1 times (1) dividing a
population of solid supports into M(N) batches, wherein M(N)
depends on N and is an integer from at least 2 to about 25; (2)
coupling the M(N) batches of solid support respectively with M(N)
different components, wherein M(N) is the number of batches during
the Nth step, said components being protected or not protected at a
group which is capable of participating in a further coupling step
and orthogonally protected at a nonparticipating group(s); (3)
adding to each batch either prior to coupling step (2),
concurrently therewith, or subsequently to step (2), from about
0.001 to about 0.5 molar equivalent of a spectrally distinguishable
fluorophore tag associated uniquely with each component in the Nth
coupling step (2), said tag being identified by its characteristic
excitation wavelength(s), emission wavelength(s), excited state
lifetime and emission intensity, said tag being activated so as to
form either a direct bond to the surface of the solid support,
either via a fuinctional group which is protected or not protected
and is the same as or different from the functional group bonded to
the component, a direct bond to the (N--I)th component, or an
indirect bond via a C.sub.1-C.sub.9 linear or branched alkyl linker
moiety which is optionally interrupted by at least one oxygen or
nitrogen atom or a carbonyl, (C--O)NH or NH(C--O) moiety, said
linker being bonded to the functional group at the surface of the
solid support, wherein when said functional group is protected,
said function group is deprotected prior to forming said direct or
indirect bond; and (4) recombining all M(N) batches and cleaving
any protecting group present at a group which is to participate in
a further coupling step; so as to form a compound having N
components; f) performing an assay capable of indicating that any
compound in the library either while bound to or cleaved from its
solid support has the property of interest; g) collecting spectral
fluorescence data for each respective solid support so as to
determine respective relative abundances of the fluorophore tags
bound thereto; and h) analyzing the collected spectral fluorescence
data by comparing the respective relative abundances of the
fluorophore tags determined in step g) so as to determine the N
components coupled in the unique reaction series for the compound,
thereby identifying the compound having the property of
interest.
38. The method of claim 37 wherein the components are independently
selected from the group consisting of an amino acid, a hydroxyacid,
an oligoamino acid, an oligopeptide, a saccharide, an
oligosaccharide, a diamine, a dicarboxylic acid, an
amine-substituted sulfhydryl, a sulfhydryl-substituted carboxylic
acid, an alicyclic, an aliphatic, a hecteroaliphatic, an aromatic
and a heterocyclic moiety.
39. The method of claim 38 wherein the saccharide is a suitably
protected D- or L-glucose, fructose, inositol, mannose, ribose,
deoxyribose or fucose.
40. The method of claim 38 wherein the oligopeptide is an
enkephalin, a vasopressin, an oxytocin, an atrial natrietic factor,
a bombesin, a calcitonin, a parathyroid hormone, a neuropeptide Y
or an endorphin, or a fragment thereof comprising at least 20% of
the components thereof, or an isosteric analogue thereof wherein
independently NH(C--O) is replaced by NH(C--O)NH, NH(C--O)O,
CH.sub.2(C--O) or CH.sub.2O; NH2, is replaced by OH, SH, N0.sub.2
CH.sub.3; CH.sub.3 S is replaced by CH.sub.3 (S--O) or CH.sub.3
CH.sub.2; indole is replaced by naphthyl or indene; hydroxyphenyl
is replaced by tolyl, mercaptophenyl or nitrophenyl; and/or
hydrogen in an aromatic ring is replaced by chlorine, bromine,
iodine or fluorine; C.sub.1-C.sub.4 alkyl is replaced by partially
or fully fluorinated Cl,-C.sub.4, alkyl.
41. The method of claim 38 wherein the oligopeptide is an ACE
inhibitor, an HIV protease inhibitor, a cytolytic oligopeptide or
an antibacterial oligopeptide.
42. The method of claim 38 wherein the aromatic is para-di
substituted benzene, biphenyl, naphthalene or anthracene, either
substituted or unsubstituted by linear or branched chain lower
alkyl, alkoxy, halogen, hydroxy, cyano or nitro.
43. The method of claim 38 wherein the heterocyclic moiety is
2,6-disubstituted pyridine, thiophene, 3,7-disubstituted
N-protected indole or 2,4-disubsituted imidazole, either
substituted or unsubstituted by linear or branched chain lower
alkyl, alkoxy, halogen, hydroxy, cyano or nitro.
44. The method of claim 37 wherein the solid support is a
microsphere, a bead, a resin or a particle, and is composed of a
material selected from the group consisting of polystyrene,
polyethylene, cellulose, polyacrylate, polyacrylamide, or
preferably a silica or glass bead.
45. The method of claim 37 wherein the solid support is chemically
modified by covalent attachment of a substituted or unsubstituted
oligo- or polyethyleneglycol, which is either terminated or
unterminated by an amine substituted by either hydroxymethyl,
chloromethyl, aminomethyl or mercaptomethyl, wherein the functional
group at the surface of the solid support is hydroxy, chlorine, NH2
or SH, respectively.
46. The method of claim 37 wherein the assay is performed while the
compound is attached to its solid support.
47. The method of claim 37 wherein the assay is performed while the
compound is cleaved from its solid support under conditions whereby
the compound remains adsorbed to the solid support.
48. The method of claim 37 wherein when the property of interest is
binding affinity of a compound to a receptor, the assay is
performed by determining a physical response to binding by a) first
admixing with the library of compounds a solution of a labelled
receptor so as to result in labelled receptor bound to at least one
compound bound to a solid support; b) removing the solution from
the solid support; and either c) washing the solid support so as
substantially to remove non-bound labelled receptor, and step (d);
or d) measuring the physical response due to bound labelled
receptor so as to determine the binding affinity.
49. The method of claim 48 wherein receptor is labelled by a
fluorescent dye, a colored dye, radioisotope or an enzyme.
50. The method of claim 48 wherein the physical response is
fluorescence emission, optical absorption or radioactivity.
51. The method of claim 37 wherein the components have a structure
independently selected from the group consisting of: ##STR3##
wherein R., R.sub.2, R.sub.3, R.sub.4,,R.sub.5, and R6 are
independently methyl, ethyl, linear or branched chain
C.sub.3-C.sub.9 alkyl, phenyl, benzyl, benzoyl, cyano, nitro, halo,
formyl, acetyl and linear or branched chain C.sub.3-C.sub.9 acyl;
wherein a, b, c, d and e are independently 0, 1, 2 or 3; wherein X,
Y and Z are independently NH, 0, S, S(--O), CO, (CO)O, O(CO),
NH(C--O) or (C--O) NH; and wherein W is independently N, 0 or
S.
52. The method of claim 37 wherein at least one component is an
amino acid, and the protected or unprotected group which is to
participate in a further coupling step is nitrogen, said protecting
group being selected from the group consisting of
N-a-fluorenylmethyloxycarbonyl, t-butyloxcarbontyl,
t-amyloxycarbonyl, (trialkysilyl) ethyloxycarbonyl, t-butyl and
benzyl;
53. The method of claim 37 wherein the fluorophore tag represents a
bit of binary code, and comprises zero, one or more than one
fluorescence dye, multiple fluorescent dyes, said dye(s) being
spectrally distinguishable by excitation wavelength, emission
wavelength, excited-state lifetime or emission intensity.
54. The method of claim 37 wherein the assay is performed by
cleaving compounds from the solid support while permitting
diffusion through solution and binding to receptors, said receptors
being arranged in proximity to each solid support.
55. The method of claim 37 wherein the fluorescence data are
collected from multiple solid supports using multi-spectral imaging
methods.
56. The method of claim 53 wherein emission intensity is
distinguished by adjusting the ratio of the relative quantities of
each fluorophore.
57. The method of claim 56 wherein the ratio is 1:1, 2:1, 3:1 or
4:1.
58. The method of claim 37 wherein the fluorophore tags are dyes
selected from the group consisting of compounds with the chemical
names: 3-(c-carboxypentyl)-3'-ethyl-oxacarbocyanine-6,6'-disulfonic
acid 1-(E-carboxypentyl)-1 '-ethyl-3
,3,3',3'-tetramethylindocarbocyanine-5,5'- disulfonic acid
1-(e-carboxypentyl)-1 '-ethyl-3 ,3 ,3',3'-tetramethyl-3H-
benz(e)indocarbocyanine-5,5',7,7'-tetrasulfonic acid
1-(E-carboxypentyl)-1
'-ethyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'- disulfonic acid
1-(F-carboxypentyl)-1 '-ethyl-3 ,3 ,3',3'-tetramethyl-3H-
benz(e)indodicarbocyanine-5,5',7,7'-tetrasulfonic acid
1-(6-carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethylindotricarbocyani-
ne-5,5'- disulfonic acid and are activated as active esters
selected from the group consisting of succinimidyl,
sulfosuccinimidyl, p-nitrophenol, pentafluorophenol, HOBt and
N-hydroxypiperidyl
59. The method of claim 37 wherein the fluorophore tags are dyes
selected from the group consisting of compounds with the chemical
names:
6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)a-
mino) hexanoic acid
6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)
amino) hexanoic acid, 6-((4,4-difluoro-1
,3-dimethyl-5-(4-methoxyphenyl)4-bora-3a,
4a-diaza-s-indacene-2-propionyl) amino)hexanoic acid,
6-(((4(4,4-difluoro-5-(2-thienyl)
4bora-3a,4a-diaza-s-indacene-3-yl) phenoxy) acetyl) amino)hexanoic
acid,
6-(((4,4-difluoro-S-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)
styryloxy)acetyl) aminohexanoic acid, and
6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)
styryloxy) acetyl)aminohexanoic acid, and are activated as active
esters selected from the group consisting of succinimidyl,
sulfosuccinimidyl, p-nitrophenol, pentafluorophenol, HOBt and
N-hydroxypiperidyl.
60. The method of claim 37 wherein the fluorophore tags are dyes
selected from the group consisting of compounds with the chemical
structures: ##STR4## and are activated as active esters selected
from the group consisting of succinimidyl, sulfosuccinimidyl,
p-nitrophenol, pentafluorphenol, HOBt and N- hydroxypiperidyl.
61. The method of claim 37 wherein one of the fluorophore tags
uniquely associated with a preselected component or reaction
comprises a ligand and a substance capable of binding specifically
to the ligand, said ligand being labelled with a fluorophore and
attached in a post-assay reaction, said substance being present on
the solid support and attached prior to, concurrently with, or
subsequent to the coupling of the component, whereby the labelled
ligand when bound to the substance indicates the presence of the
preselected component.
62. The method of claim 37 wherein the solid support is a bead, and
spectral fluorescence data are collected by a) forming either a
static planar array or a dynamic planar array of beads; and b)
obtaining a fluorescence image for at least one bead.
63. The method of claim 62 wherein the planar array of beads is
formed adjacent to the planar walls of a sandwich flow cell and
controlled by light-controlled electrokinetic means.
64. The method of claim 62 wherein the dynamic planar array of
beads is formed by using an apparatus capable of dynamically
assembling and disassembling an array of beads at an interface
between an electrode and an electrolyte solution, said apparatus
comprising: i) an electrode, an electrolyte solution and an
interface there between; ii) a plurality of beads located in said
electrolyte solution; iii) said electrode being patterned to
include at least one area of modified electrochemical properties;
iv) an illumination source which illuminates said electrode with a
predetermined light pattern; v) an electric field generator which
generates an electric field at said interface to cause the assembly
of an array of beads in accordance with the predetermined light
pattern and the electrochemical properties of said electrode; and
vi) an electric field removal unit which removes said electric
field to cause the disassembly of said array of beads.
65. The method of claim 62 wherein spectral fluorescence data are
collected for the bead array by initially forming a spatially
encoded array of beads suspended at an interface between an
electrode and an electrolyte solution, comprising the following
steps: i) providing an electrode and an electrolyte solution; ii)
providing multiple types of particles, each type being stored in
accordance with chemically or physically distinguishable particle
characteristics in one of a plurality of reservoirs, each reservoir
containing a plurality of like-type particles suspended in said
electrolyte solution; iii) providing said reservoirs in the form of
an mxn grid arrangement; iv) patterning said electrode to define
mxn compartments corresponding to said mxn grid of reservoirs; v)
depositing mxn droplets from said mxn reservoirs onto said
corresponding mxn compartments, each said droplet originating from
one of said reservoirs and remaining confined to one of said mxn
compartments and each said droplet containing at least one
particle; vi) positioning a top electrode above said droplets so as
to simultaneously contact each said droplet; vii) generating an
electric field between said top electrode and said mxn droplets;
viii) using said electric field to form a bead array in each of
said nan compartments, each said bead array remaining spatially
confined to one of said man droplets; ix) illuminating said mxn
compartments on said patterned electrode with a predetermined light
pattern to maintain the position of said bead arrays in accordance
with said predetermined light pattern and the pattern of mxn
compartments; and x) positioning said top electrode closer to said
electrode thereby fusing said mxn droplets into a continuous liquid
phase, while maintaining each of said mxn bead arrays in one of the
corresponding mxn compartments.
66. The method of claim 65 wherein said compartments are
hydrophilic and the remainder of said electrode surface is
hydrophobic.
67. The method of claim 37 wherein N is an integer from at least 3
to about 12.
68. The method of claim 37 wherein M and M(N) are independently an
integer from at least 4 to about 12.
69. The method of claim 37 wherein from about 0.01 to about 0.05
molar equivalent of a spectrally distinguishable fluorophore tag is
added in step c).
70. A compound having a selected property of interest as identified
in accord with claim 37.
71. A chemical library prepared in accord with claim 37.
72. An apparatus for identifying a compound having a selected
property of interest in a library of compounds, each of said
compounds being bound to its respective solid support, and being
produced by a unique reaction series composed of N coupling and
reaction steps, wherein each said compound is prepared from a set
of components which are independently the same or different, and N
is an integer from at least 1 to about 100, said solid support
being at least one particle array, said apparatus comprising: a) an
electrode and an electrolyte solution having an interface
therebetween; b) an electric field generator which generates an
electric field at an interface between an electrode and an
electrolyte solution; c) said electrode being patterned to modify
the electrochemical properties of said electrode; d) an
illuminating source which illuminates said interface with a
predetermined light pattern to control the movement of said
particles in accordance with said predetermined light pattern and
the electrochemical properties of said electrode; e) means for
preparing said chemical library, which comprises: i) means for
dividing a population of solid supports having at least one type of
a first finctional group at the surface of said solid support
selected from the group consisting of CO.sub.2H, OH, SH, NH2, NHR,
CH.sub.2Cl, CH.sub.2Br and CHN.sub.2, wherein R is a linear
C.sub.1-C.sub.9 alkyl group, into M batches, wherein M is an
integer from at least 2 to about 50; ii) means for coupling the M
batches of solid support in a set of at least one reaction
respectively with M different initial components so as to form a
bond with the solid support via said first fuinctional group, said
components being protected or unprotected at a group which is to
participate in a further coupling step and orthogonally protected
at non-participating group(s); iii) means for adding to each batch
either prior to coupling step ii), concurrently therewith, or
subsequently to step ii), from about 0.001 to about 0.5 molar
equivalent of a spectrally distinguishable fluorophore tag
associated uniquely with each initial component, said tag being
identified by its characteristic excitation wavelength(s), emission
wavelength(s), excited state lifetime and emission intensity, said
tag being activated so as to be capable of forming either a direct
bond to the surface of the solid support, either via the first or a
second functional group which is protected or unprotected and is
the same as or different from said first functional group bonded to
the component, or an indirect bond via a C.sub.1-C.sub.9, linear or
branched alkyl linker moiety which is either interrupted or
uninterrupted by either at least one oxygen or nitrogen atom or a
carbonyl, (C--O)NH or NH(C--O) moiety, said linker being bonded to
said second functional group at the surface of the solid support,
wherein when said second functional group is protected, said second
functional group is deprotected prior to forming said direct or
indirect bond; and either iv) means for recombining all M batches
and cleaving any protecting group present at a group which is to
participate in a further coupling step, and steps v)-viii); or v)
means for iteratively N-1 times (1) dividing a population of solid
supports into M(N) batches, wherein M(N) depends on N and is an
integer from at least 2 to about 25; (2) coupling the M(N) batches
of solid supports respectively with M(N) different components,
wherein M(N) is the number of batches during the Nth step, said
components being protected or unprotected at a group which is
capable of participating in a further coupling step and
orthogonally protected at a non-participating group(s); (3) adding
to each batch either prior to coupling step (2), concurrently
therewith, or subsequently to step (2), from about 0.001 to about
0.1 molar equivalent of a different spectrally distinguishable
fluorophore tag associated uniquely with each component during the
Nth coupling step (2), said tag being uniquely identified by its
excitation wavelength, emission wavelength, excited-state lifetime
or emission intensity, whereby said tag is activated so as to be
capable of forming either a direct bond to the solid support,
either via an Nth functional group which is protected or
unprotected and is the same as or different from the first
finctional group, or an indirect bond thereto via a C.sub.1-C.sub.9
linear or branched alkyl linker moiety which is either interrupted
or uninterrupted by either at least one oxygen or nitrogen atom or
a carbonyl or NH(C--O) moiety, or a direct bond to the (N--I)th
component which if protected is priorly deprotected, said tag or
linker being bound via the group which is to participate in a
further coupling step, wherein when said Nth functional group is
protected, said Mh functional group is deprotected prior to forming
said direct or indirect bond; and (4) recombining all M(N) batches
and cleaving the protecting group present if present at a group
which is to participate in a further coupling step; so as to form a
compound having N components; vi) means for performing an assay
capable of indicating that any compound in the library either while
bound to or cleaved from its solid support has the property of
interest; vii) means for collecting spectral fluorescence data for
each respective solid support so as to determine respective
relative abundances of the fluorophore tags bound thereto; viii)
means for analyzing the collected spectral fluorescence data by
comparing the respective relative abundances of the fluorophore
tags determined in step vii) so as to determine the N components
coupled in the unique reaction series for the compound, thereby
identifying the compound having the selected property of interest.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to the field of
analytical chemistry.
[0002] The present invention specifically relates to a highly
parallel mode of presenting and probing multiple chemical
compounds, with applications to combinatorial library synthesis,
ultrahigh-throughput screening, diagnostic assays for multiple
agents and sensors. The present invention introduces several color
codes to label collections of carrier particles such as colloidal
beads; in addition, the present invention describes a method and
apparatus for the in-situ interrogation of beads or collections of
beads by way of multi-color fluorescence imaging and spectral
analysis of individual beads to ascertain the chemical identities
of bead-anchored compounds. The encoding of beads by simple and
extended simple color codes and by binary and extended binary color
codes may be augmented by measuring bead size and shape or other
physico-chemical properties such as polarizability embedded in the
bead core.
BACKGROUND OF THE INVENTION
1-Solid Phase Chemical Libraries
[0003] An emerging paradigm for lead discovery in pharmaceutical
and related industries such as agricultural biotechnology, is the
assembly of novel synthetic compound libraries by new methods of
solid state "combinatorial" synthesis. Combinatorial chemistry
refers to a set of strategies for the parallel synthesis and
testing of multiple compounds or compounds mixtures, either in
solution or in solid supports in the form of beaded resins
("beads"). In general, a combinatorial synthesis employing M
precursors in each of N reaction steps produces M N compounds. For
example, a combinatorial synthesis produces 4 N oligon-nucleotides
in N steps, each employing 4 oligonucleotide precursors; similarly,
a combinatorial synthesis of N steps, each employing 20 amino acid
precursors, produces 20 N oligopeptides.
1.1--One Bead/One Compound Chemical Libraries
[0004] One implementation of combinatorial synthesis that is
suitable to produce very large chemical libraries relies on solid
supports in the form of beaded resins ("beads") and encodes
reaction steps in a "divide, couple and recombine" (DCR) strategy
(FIG. 1), also refereed to as "resin-splitting" synthesis. The
resulting "one bead/one compound" chemical libraries contain from
10 6 to 10 8 compounds. These libraries are screened by performing
a wide variety of chemical and biochemical assays to identify
individual compounds eliciting a positive response. The chemical
identity of such compounds can be determined by direct
analysis.
[0005] Two methods of direct analysis are micro-sequencing and mass
spectrometry. Both methods require the physical isolation of
synthesis beads displaying compounds of interest and both require
off-line chemical analysis based on substantial amounts of
compound--tens to hundreds of picomoles. Micro-sequencing, limited
to libraries of oligopeptides and oligonucleotides, does not
distinguish between stereoisomers. Mass spectrometry is unable to
distinguish between precursors of equal mass such as D- and L-amino
acids or leucine and isoleucine. The requirement of direct chemical
analysis for a substantial quantity of compound dictates the use of
large bead resins (a typical bead diameter is 130 .mu.m) to ensure
that picomolar quantities of each compound can be recovered, even
when it is becoming increasingly desirable to perform high
throughput screening of the compound library in miniaturized
environments to reduce requisite volumes of sample and reagents and
to enhance throughput.
1.2--Encoded One Bead/One Component Chemical Libraries
[0006] One approach to overcoming the serious limitations of
standard one bead/one compound chemical libraries is to encode
chemical compound identities. This facilitates the identification
of compounds not amenable to direct determination by
micro-sequencing or mass spectrometry. One encoding method employs
the co-synthesis of peptides and oligonucleotides to represent the
identity of non-sequenceable synthesis products (Nikolaiev et al.,
"Peptide-Encoding for Structure Determination of Non-Sequenceable
Polymers Within Libraries Synthesized and Tested on Solid-Phase
Supports", Peptides Res. 6, 161 (1993), the contents of which are
included herein by reference). A second method, compatible with a
wider range of chemical reaction conditions, employs a set of
tagging molecules to record the reaction histories of beads.
[0007] One implementation of the latter method uses a set of
pre-synthesized, chromatographically distinguishable molecular tags
T1, T2, . . . , TM to construct a chemical binary code. In prior
art, molecular tags are structurally related molecules (FIG. 2)
which can be identified by their characteristic gas chromatographic
retention times (Still et al., "Complex combinatorial libraries
encoded with tags", U.S. Pat. No. 5,565,324, the contents of which
are included herein by reference).
[0008] At each step of DCR synthesis, a unique tag from the set is
added to each divided aliquot to record the reaction carried out
with that aliquot. The concept may be illustrated by examining the
steps of a 2-step synthesis using reagents R.sup.1.sub.1,
R.sub.2.sup.1 and R.sub.3.sup.1 in step 1, and reagents
R.sup.2.sub.1, R.sup.2.sub.2 and R.sup.2.sub.3 in step 2, to
generate nine products. The reagents of the first step are uniquely
identified by the binary addresses 01 (R.sup.1.sub.1), 10
(R.sup.1.sub.2) and 111 (R.sup.1.sub.3), and the reagents of the
second step are uniquely identified by the binary addresses
01(R.sup.2.sub.1), 10(R.sup.2.sub.2) and 11(R.sup.2.sub.3). Each
binary address is chemically represented in terms of a set of
molecular tags: T1 (01 in step 1 representing R.sup.1.sub.1), T2
(10 in step 1 representing R.sup.1.sub.3) and T2T1 (11 in step 1
representing R.sup.1.sub.3) and analogously with T3 (01 in step 2
representing R.sup.2.sub.1), T4 (10 in step 2 representing
R.sup.2.sub.2) and T4T3 (11 in step 2 representing
R.sup.2.sub.3).
[0009] A sequence of reaction steps is recorded by simply
concatenating binary addresses. Thus, 11.01, read right to left,
would indicate the sequence "reagent R.sup.2.sub.3 in step 2,
reagent R.sup.1.sub.1 in step 1". The chemical representation of
this sequence is T4T3.T1, and the presence on the bead of this
particular set of tags indicates the chemical identity of the
bead-anchored synthesis product. The strategy is readily
generalized to larger reactions. For example, 7 reagents to be used
in each reaction step can be uniquely identified by the binary
addresses 001 (R.sup.1.sub.1), 010 (R.sup.1.sub.2), . . . , 111
(R.sup.1.sub.7). Although superior to un-encoded one bead/one
compound methods, nevertheless the tagging strategy of prior art
still suffer from three limitations. First, individual beads of
interest must be physically isolated from the rest; next, molecular
tags must be chemically or photochemically cleaved from the bead
and cleaved tags must be collected; and finally, chemical analysis
(e.g., gas chromatography) must be performed. These numerous
time-and labor-intensive manipulations eliminate much of the
enhancement in throughput gained by the DCR synthesis strategy.
1.3 Screening and Lead Compound Optimization
[0010] The high specificity of typical biological substrate-target
interactions implies that the vast majority of compounds in a
library will be inactive for any particular target. Thus, the task
of screening is to identify the very few compounds within the
library that display activity in binding or in functional assays.
Common targets include enzymes and receptors as well as nucleic
acids.
[0011] To implement the rapid screening and scoring of an entire
library of synthetic compounds, in practice containing 10 4 to 10 8
compounds, requires systematic screening procedures if the task is
to be completed within viable time frames. Several assay formats
have been described to implement the screening of bead-based
combinatorial libraries. These include: reaction of a collection of
beads, allowed to settle under gravity, with an enzyme-labeled or
fluorophore-labeled target molecule followed by visual detection
(Lam et al., "A new type of synthetic peptide library for
identifying ligand-binding activity", Nature 354 (1991), the
contents of which are included herein by reference); incubation of
beads with radio-labeled target molecules and subsequent agarose
immobilization of beads and auto-radiographic detection (Kassaj
ian, Schellenberger and Turck, "Screening of Synthetic Peptide
Libraries with Radio-labeled Acceptor Molecules", Peptide Res. 6,
129 (1993), the contents of which are included herein by
reference); and partial release of compounds from beads for
solution-phase testing (Salmon et al., "Discovery of biologically
active peptides in random libraries: Solution-phase testing after
staged orthogonal release from resin beads", Proc. Natl. Acad. Sc.
USA 90, 11708 (1993), the contents of which are included herein by
reference).
[0012] WO95/32425 provides a method of preparing combinational
libraries using a method of encoding combinational libraries with
fluorophore labeled beads. According to the method, a first
combinational library is prepared by conducting a set of reactions
on tagged beads to afford an encoded first registry (i.e., step in
the synthetic sequence). A second combinational library is prepared
using similar reaction steps but the tagged beads are combined and
separated prior to the first reaction sequence and the beads are
sorted prior to the second reaction sequence. Subsequent libraries
are prepared as for the second library except that the sorting step
takes place prior to a different registry in each subsequent
library. Thus. WO95/32425 teaches only individually labelling the
first step and physical separatois of beads to identify each
modified combinational library.
[0013] Nederlof et al., Cytometry, 13, 839-845 (1992), teaches the
use of ratio labeling as a way of increasing the number of
simultaneously detectable probes beyond the seven used previously.
In this approach, ratio-labelled probes are identified on the basis
of the ratio of color intensity, not just the particular colors
used. Fluorescence ratios are measured and used as additional
encoding colors. The method requires double-labeling of probes
using different ratios of labels. The method is not specifically
directed to synthetic combinational libraries. Accordingly, the
field of Nederlof's method is the detection of multiple DNA/RNA
sequence by in situ hybridization, and is not relevant to the field
of encoding of synthetic chemical libraries.
[0014] Speiche, Ballard & Ward, Nature Genetics, 12, 368
(1996), describe a method of characterizing complex chromosomal
karyo types using multi-fluorescence in situ hybridization. Instead
of using ratio-double labelling as in Nederlof, Speiche et al. use
a set of six fluorescent dyes with spectral emission peaks spread
across the photometric response range to visualize 27
combinationally labelled probes. Speiche et al. do not disclose a
method of encoding synthetic combinational libraries.
[0015] Still et al., Proc. Nat'l Acad. Sci., 90, 10922-926 (1993),
disclose a method of synthesis of tagged combinational libraries
using a binary code based on different electrophoric tags. The
method requires use of photocleavable molecular tags which comprise
variously substituted aryl moieties linked via a variable-length
aliphatic hydrocarbon chain, whereby the tags when cleaved are
distinctly resolvable by capillary gas chromatography with
electochemical detection. Color detection is not used in this
method. The method also requires cleavage from the solid support in
order to analyze the sequence. In related work, Still et al. U.S.
Pat. No. 5,721,099 disclose methods of preparing encoded
combinatorial libraries, but again the method requires cleavage of
the identifier tags prior to analysis of the encoded reaction
history. In contrast, the present invention provides an in situ
approach to the interrogation of encoded combinatorial libraries,
and represents an advance over the prior methods of encoding
libraries. The success of the present invention is unexpected in
view of the prior approaches because of the scattering phenomena
expected for a spectral analysis performed in heterogeneous media
which would dissipate spectral signal-to-noise giving rise to
practical difficulties in detecting accurately relative abundance
information for fluorophore tags. The present methodology
demonstrates for the first time a way of solving these practical
problems in performing in situ encoding and interrogation of
combinatorial libraries.
II--Multi-Agent Monitoring and Diagnostics
[0016] Diagnostic panels display multiple chemistries to screen
unknown solutions for the presence of multiple agents. For example,
blood group specificity is determined by spotting an unknown blood
sample onto a panel of surface-bound antibodies whose arrangement
in the panel reflects their antigen-specificity. Antigen-binding to
any specific patch in the panel reveals the chemical identify of
the antigen and enhance the blood type. Another realization of the
same concept of displaying multiple diagnostic probes in a
spatially encoded panel or array involves screening of mutations by
assaying for hybridization of DNA to one of a large number of
candidate matching strands which are placed in known positions on a
planar substrate in a checkerboard pattern. This may be achieved by
dispensing droplets containing distinct probes, or may involve the
in-situ synthesis of oligonucleotide strands of varying
composition.
[0017] Spatial encoding relies on the panel or array fabrication
process to preserve chemical identity, adding time and expense. As
the number of fields in the checkerboard increases, so does the
challenge of fabricating the requisite array. In addition, probes
must be immobilized--usually by adhesion to the surface of a planar
substrate--to maintain the integrity of the spatial encoding
scheme. In practice, this assay format can be problematic: sample
accumulation can be slow and probe accessibility restricted.
III--Current Applications of Multicolor Fluorescence Detection
[0018] The present invention describes a method and apparatus for
in-situ interrogation and deconvolution of bead-based combinatorial
libraries using multi-color fluorescence imaging and spectral
analysis. Recent applications of multi-color fluorescence
spectroscopy to DNA sequencing and chromosome painting place
requirements on sensitivity and wavelength selectivity exceeding
those encountered in conventional applications such as
determinations of fluorescence intensity ratios.
[0019] Within the context of DNA sequencing, a variety of
configurations for rapid detection of 4-color fluorescence have
been described. These involve: a dedicated photomultiplier tube
detector for each emission wavelength, with corresponding sets of
beam splitters in the optical path to produce spatially separated
beams; a single detector and rotating filterwheel to select the
desired set of wavelengths in a multiplexed recording mode; or a
dispersive arrangement that relies on a prism or grating to split
the emitted light from multiple fluorophores according to
wavelength and takes advantage of recent advances in charge-coupled
device (CCD) technology to record spectra on an integrating linear
of rectangular CCD array (Karger et al., "Multiwavelength
fluorescence detection for DNA sequencing using capillary
electrophoresis", Nucl. Acids Res. 19, 4955 (1991), the contents of
which are incorporated herein by reference).
SUMMARY OF THE INVENTION
[0020] The present invention provides a method to construct several
color codes for the purpose of uniquely labeling members of a group
of beads or equivalent objects ("beads") to preserve the chemical
identity of the beads and thus the identity of bead-coupled
chemical compounds. These color codes are based on a set of
encoding fluorophores of distinguishable wavelengths, excited-state
lifetimes and levels of intensity, the latter controlled by
adjusting the abundances of dyes. Specifically, the present
invention describes a method and apparatus for the encoding and
in-situ interrogation of a set of distinct, bead-based
chemistries.
[0021] Binary and extended binary color codes offer large coding
capacity and represent a general strategy to encode multi-step
reaction histories such as those encountered in
divide-couple-recombine (DCR) synthesis strategies for
combinatorial chemical libraries, as illustrated and discussed
herein.
[0022] Simple and extended simple color codes offer an efficient
strategy to encode a smaller set of distinct chemistries that are
typical of panels displaying multiple targets or probes in
biochemical assays including multi-agent diagnostic and
environmental tests and other biochemical assays.
[0023] All color codes can be augmented by varying distinguishable
features of beads such as shape and size or other suitable
physico-chemical parameter associated with bead cores such as
polarizability.
[0024] The identity of the compound anchored to any specific bead
is determined in-situ by optically probing individual beads to read
the color code, as descried herein. This ensures the identification
of bead-anchored chemical compounds without the need for physical
separation and without the need for off-line chemical analysis.
[0025] The encoding strategy of the present invention is compatible
with all formats of bead-based combinatorial synthesis and
screening described to date. A preferred implementation that has
the advantage of enabling miniaturization and automation of
screening and decoding operations relies on planar bead arrays
which may be formed, maintained and manipulated adjacent to a
planar electrode surface.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Other objects, features and advantages of the invention
discussed in the above brief explanation will be more clearly
understood when taken together with the following detailed
description of an embodiment which will be understood as being
illustrative only, and the accompanying drawings reflecting aspects
of that embodiment, in which:
[0027] FIG. 1 is an illustration of "Divide-Couple-Recombine"
combinatorial synthesis; FIG. 2 is an illustration of labeling
individual synthesis beads with chemical tags ("bar codes").
Examples of molecular structures used for such tags are also shown:
different tags are made by varying n and Ar;
[0028] FIG. 3 is an illustration of two alternative methods of
placing fluorophore or chromophore tags (F) on synthesis beads;
FIG. 4 is an illustration of binary color coding with fluorophores,
Y, B, G and R. The example enumerate coded bead populations
produced in combinatorial peptide synthesis employing reagents
R.sup.1.sub.1, R.sup.1.sub.2, R.sup.1.sub.3 and R.sup.1.sub.4 in
step 1 and reagents R.sup.2.sub.1, R.sup.2.sub.2, R.sup.2.sub.3 and
R.sup.2.sub.4 in step 2 (see also: Table I);
[0029] FIG. 5 is an illustration of emission spectra of the CyDye
family of commercially available fluorescent dyes whose spectral
characteristics are summarized in the table accompanying the figure
(Amersham LIFE SCIENCE, Catalog of Multicolor Fluorescent Reagents,
1995, the contents of which are included herein by reference);
[0030] FIG. 6 is an illustration of a random bead array encoded
according to the simple color code SCC(1=1, m=5);
[0031] FIG. 7 is an illustration of a multi-color fluorescence
microscope with integrated spectral analysis based on dispersive
optics;
[0032] FIG. 8 is an illustration of several geometries of
multi-color fluorescence imaging and spectrometry.
[0033] FIG. 9 is an illustration of an example of a solid support
having a hydroxy functional group at its surface which is modified
by a linker which is formed in a multistep process involving a
deprotection of an MNmt protecting group and subsequent reaction
with an activated ester of a fluorescent dye in accord with the
present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Implementation of Color Codes
[0034] The color coding strategy of the present invention provides
a method to place a set of fluorophores--or, more generally,
chromophores--on each bead so as to uniquely encode the chemical
identity of the compound on that bead. Specifically, during each
coupling step in the course of DCR combinatorial synthesis, one or
more fluorophores are attached to each bead. Decoding is based on
the determination of relative abundances of fluorophores on a bead
of interest by in-situ optical interrogation.
[0035] Fluorophores can be added in two ways. In the first method,
the fluorophore is added directly to a small fraction of the
nascent compound, thereby terminating further synthesis of that
fraction of nascent compound (FIG. 3A). In the second method, the
label is covalently attached to reserved reaction sites other than
nascent compound to ensure that precursors are not terminated by
labeling (FIG. 3B). In the first method and in most implementations
of the second method, the quantity, x, of flurophore added to each
bead is sub-stoichiometric with respect to nascent compound, with x
typically in the range 0.001 to 0.1 mole equivalents of nascent
compound on the bead. Three factors govern the choice of x. First,
the density of tags on beads must not materially interfere with
synthesis and with subsequent screening assays. Second, the density
of tags on beads must remain sufficiently low as to avoid
complication due to fluorescence energy transfer. Third, labeled
sites must be present in sufficient number to meet the requirements
of signal detection and discrimination, as discussed herein.
[0036] To implement the color coding strategy, the present
invention takes advantage of three properties of fluorophores to
construct an alphabet of fluorophore tags, namely: emission
wavelength; excited-state lifetime, and emission intensity.
Denoting by m.sub.F the number of available fluorophores with
distinguishable emission maxima and/or excited state lifetimes, and
denoting by m.sub.I the number of distinguishable intensity levels,
controlled by adjusting relative quantities of fluorophores (e.g.
x, 2.times., 3.times. . . . ), the size of the alphabet of
fluorophore tags is m=m.sub.F*m.sub.I. The surfaces of labeled
beads will display a multiplicity of distinct fluorophores (see
FIG. 4). In-situ optical interrogation of these multi-colored beads
serves to record emission spectra from which relative abundances of
fluorophores are determined to decipher the color code, as
discussed and illustrated herein.
Binary Color Codes
[0037] One rendition of this code is a binary color code (BCC)
using m.sub.F fluorophores, all with m.sub.1=1. This BCC will
encode up to 2 m.sub.F distinct compounds. In this BCC, the m
fluorophores could differ in excite-state lifetimes, emission
maxima or both. For convenience, the following specific example
uses fluorophores differing solely in their emission maxima
("colors"). The combinatorial synthesis of 16 products in two
reaction steps, each using a set of N=4 reagents, would be encoded
as follows: TABLE-US-00001 TABLE I Step 1: R.sup.1.sub.1 (00) No
color R.sup.1.sub.2(01) Red R.sup.1.sub.3(10) Green
R.sup.1.sub.4(11) Red + Green Step 2: R.sup.2.sub.1(00) No color
R.sup.2.sub.2(01) Blue R.sup.2.sub.3(10) Yellow R.sup.2.sub.4(11)
Yellow + Blue R.sup.2.sub.1, R.sup.1.sub.1 00.00 NN.NN no color
R.sup.2.sub.3, R.sup.1.sub.1 10.00 YN.NN Y R.sup.2.sub.1,
R.sup.1.sub.2 00.01 NN.NR R R.sup.2.sub.3, R.sup.1.sub.2 10.01
YN.NR YR R.sup.2.sub.1, R.sup.1.sub.3 00.10 NN.GN G R.sup.2.sub.3,
R.sup.1.sub.3 10.10 YN.GN YG R.sup.2.sub.1, R.sup.1.sub.4 00.11
NN.GR GR R.sup.2.sub.3, R.sup.1.sub.4 10.11 YN.GR YGR
R.sup.2.sub.2, R.sup.1.sub.1 01.00 NB.NN B R.sup.2.sub.4,
R.sup.1.sub.1 11.00 YB.NN YB R.sup.2.sub.2, R.sup.1.sub.2 01.01
NB.NR BR R.sup.2.sub.4, R.sup.1.sub.2 11.01 YB.NR YBR
R.sup.2.sub.2, R.sup.1.sub.3 01.10 NB.GN BG R.sup.2.sub.4,
R.sup.1.sub.3 11.10 YB.GN YBG R.sup.2.sub.2, R.sup.1.sub.4 01.11
NB.GR BGR R.sup.2.sub.4, R.sup.1.sub.4 11.11 YB.GR YBGR
[0038] The binary representation of four reagents is R.sub.1(00),
R.sup.1.sub.2 (01), R.sup.1.sub.3 (10) and R.sup.1.sub.4 (11) for
the reagents used in step 1, and R.sup.2.sub.1(00),
R.sup.2.sub.2(01), R.sup.2.sub.3(10) and R.sup.2.sub.4(11) for
those in step 2. As before, sequences of reaction steps correspond
to concatenated binary codes, and in the example all 4 2=16
possible sequences are represented by 4-bit strings. Thus, the
sequence: "reagent R.sup.2.sub.3 in step 2, reagent R.sup.1.sub.4
in step 1" would be represented by the string 10.11 (read right to
left). Using an alphabet of four fluorophores, with colors denoted
by R, G, B, and Y as before, and assigned (Y, B, G, R) to represent
4-bit strings, the 2 4 possible strings (read right to left) are
encoded in BCC (m=4) as displayed in table I and in FIG. 4.
[0039] A second rendition of the color code is a binary color code
using m.sub.F fluorophores with varying relative abundances and
thus varying intensities at each step. The resulting eXtended
binary color code (XBCC) will encode 2 (m.sub.F{dot over (
)}m.sub.I) distinct compounds. For example, using an alphabet (2G,
2R, G, R) with only two distinct colors to represent 4-bit strings,
2 4 possible strings (read right to left) are encoded in XBCC
(m.sub.F=2, m.sub.1=2) as enumerated in Table II. In the example,
deconvolution will require discrimination of four distinct
intensity levels for each of the two emission bands. If N steps are
involved, the number of intensity levels to be discriminated in the
extended binary color code XBCC (M.sub.F, m.sub.I) may be as high
as N{dot over ( )}m.sub.I. The attainable intensity discrimination
is ultimately limited by the signal-to-noise ratio attainable in
the spectral analysis of individual beads. TABLE-US-00002 TABLE II
Step 1: R.sup.1.sub.1 (00) No color R.sup.1.sub.2(01) Red
R.sup.1.sub.3(10) Green R.sup.1.sub.4(11) Red + Green Step 2:
R.sup.2.sub.1(00) No color R.sup.2.sub.2(01) 2Red R.sup.2.sub.3(10)
2Green R.sup.2.sub.4(11) 2Red + 2Green R.sup.2.sub.1, R.sup.1.sub.1
00.00 NN.NN no color R.sup.2.sub.3, R.sup.1.sub.1 10.00 2GN.NN GG
R.sup.2.sub.1, R.sup.1.sub.2 00.01 NN.NR R R.sup.2.sub.3,
R.sup.1.sub.2 10.01 2GN.NR GGR R.sup.2.sub.1, R.sup.1.sub.3 00.10
NN.GN G R.sup.2.sub.3, R.sup.1.sub.3 10.10 2GN.GN GGG
R.sup.2.sub.1, R.sup.1.sub.4 00.11 NN.GR GR R.sup.2.sub.3,
R.sup.1.sub.4 10.11 2GN.GR GGGR R.sup.2.sub.2, R.sup.1.sub.1 01.00
N2R.NN RR R.sup.2.sub.4, R.sup.1.sub.1 11.00 2G2R.NN GGRR
R.sup.2.sub.2, R.sup.1.sub.2 01.01 N2R.NR RRR R.sup.2.sub.4,
R.sup.1.sub.2 11.01 2G2R.NR GGRRR R.sup.2.sub.2, R.sup.1.sub.3
01.10 N2R.GN RRG R.sup.2.sub.4, R.sup.1.sub.3 11.10 2G2R.GN GGGRR
R.sup.2.sub.2, R.sup.1.sub.4 01.11 N2R.GR RRRG R.sup.2.sub.4,
R.sup.1.sub.4 11.11 2G2R.GR GGGRRR
[0040] Another example describes the color-coding of products
created in a combinatorial synthesis using 7 reagents in the first
step, 6 reagents in each of the final two steps.
[0041] Reagents are represented by binary addresses R1(001),
R2(010), R3(011) . . . , R7(111); for simplicity of notation, we
omit the superscript for reagents (R) used in different steps.
[0042] Let m.sub.F=4 (color denoted as before) and m.sub.I=2. The
following XBCC based on an 8-letter alphabet (2Y, 2B, 2G, 2R, Y, B,
G, R) and illustrated in Table III may be devised to encode the
7{dot over ( )}6{dot over ( )}6=252 synthesis products created in
this synthesis. While the construction of the XBCC would require
9-bit strings to represent the full set of 8 3=512=2 9
configurations created by all possible concatenations of 3-bit
strings, the actual 252 required configurations of the example can
in fact be accommodated in the set of 2 8 possible 8-bit strings by
making replacements of the sort indicated in the example. Thus, the
reaction sequence "reagent 6 in step 3, reagent 1 in step 2,
reagent 3 in step 1" is represented by the XBCC (m.sub.F=4,
m.sub.I=2) as follows (read right to left):
R6.R1.R3=2.times.2B.N.G=2G2RY.N.G and thus corresponds to GGGRRY.
TABLE-US-00003 TABLE III R1 R2 R3 R4 R5 R6 R7 000 001 010 011 100
101 110 Step1(7) N R G GR B BR BG NOT USED: BGR Step2(6) N Y 2R 2RY
2G 2GY NOT USED: 2G2R, 2G2RY Step3(6) N 2B 2Y 2Y2B 2X 2X2B Note: By
convention, make the following replacements: 2X <- 2G2R, 2X2B
<- 2G2RY
Simple Color Codes
[0043] In contrast to the complex task of encoding reaction
histories in a multi-step combinatorial synthesis, many
applications require the distinction of only a limited set of
chemistries. Simple color codes (SCC) can be constructed for this
purpose. While not matching the encoding capacity of the
corresponding binary color codes, these color codes are entirely
suitable in many instances in which the chemical distinctions of
interest are created in a single reaction step, such as the
coupling of a diagnostic probe to a bead. Examples of such limited
chemical complexity include sensing applications as well as
multi-agent monitoring and diagnostics.
[0044] As with binary color codes, the construction of simple color
codes takes advantage of distinguishable wavelengths, lifetimes and
intensities of available fluorophores. A general version of the SCC
based on a total of m fluorophores is constructed by using equal
amounts of 1 flurophores to encode each distinct chemical species
of interest, where 1.ltoreq.1.ltoreq.m. In this code, the set of
possible combinations of colors is equivalent to the number of
possible configurations, S_r(1,m), of a sample of size 1 drawn with
replacement from a reservoir of m, S_R(1,m)-(m+1-1)!/1!(m-1)!.
Replacement allows for multiple instances of one color in each
string.
[0045] For example, if 4 distinct fluorophores (m=4) were
available, and combinations of 3 (1=3) were used--in equal relative
abundances--for each distinct chemical species of interest, the
generalized SCC would provide a total of 20 distinct
configurations. These are listed in table IV, denoting by R, G, B
and Y the colors in a 4-color alphabet. Thus, the SCC (1=3, m=4)
will uniquely encode the products generated in a single step of
coupling up to 20 distinct antibodies to carrier beads; each of 20
reaction vessels would receive a mixture of three fluorophores in
accordance with the set listed Table IV. The presence of several
known fluorophores provides the basis to invoke coincidence methods
to detect and monitor weak signals and so to enhance assay
sensitivity. TABLE-US-00004 TABLE IV (R, R, R) (G, G, G) (B, B, B)
(Y, Y, Y) (R, R, G) (G, G, B) (B, B, Y) (R, R, B) (G, G, Y) (R, R,
Y) (R, G, G) (G, B, B) (B, Y, Y) (R, G, B) (G, B, Y) (R, G, Y) (R,
B, B) (G, Y, Y) (R, B, Y) (R, Y, Y)
[0046] EXtended simple color codes (XSCC) can be constructed by
varying relative abundances of fluorophores to create a set of
distinguishable intensity levels for each of the fluorophore
species in the alphabet. As with the XBCC, the XSCC permits control
of m.sub.I intensity levels for each of m.sub.F florophore species
in the alphabet.
[0047] Particularly easy to realize is the special case of SCC and
XSCC where l=1; only a single fluorophore marks each chemical
species of interest.
Further Enhancements
[0048] All color codes previously discussed herein can be further
augmented by varying certain physico-chemical parameters of beads.
For example, the number of encoded configurations may each be
attached to a set of beads whose respective shapes, mean sizes,
polarizabilities or other physico-chemical properties differ
sufficiently so as to be distinguishable. By using S distinct sets
of beads, the number of encoded configurations represented with
XBCC(m) is increased to S*2 m.
[0049] BCC and XBCC encode chemical compound identity in terms of
the relative abundances of fluorophores coupled to each bead.
Accordingly, all permutations of a string of fluorophore tags are
equivalent because they result in the same relative abundances.
However, it has not escaped our notice that the implementation of
the color code in which labeling leads to compound termination (see
FIG. 3A) also retains a record of the order in which different
color labels were added to each bead. Consequently, the analysis of
molecular weights of labeled compounds will reveal the order in
which labeling occurred.
Chemical Realization of Extended Binary Color Code
[0050] The realization of a chemical color code relies on a set
("alphabet") of chemically activated fluorophores with minimally
overlapping absorption and emission spectra. We discuss here the
case of the Extended Binary Color Code; other codes may be realized
in analogous fashion. Although the implementation of a color code
according to the present invention is illustrated herein by way of
a specific family of fluorophores, the method is equally suitable
for implementation with other fluorophores and chromophores whose
distinctive spectral features serve to construct an alphabet of
tags as described herein. An example of a suitable alphabet of six
colors is provided by the CyDye(TM) family of indocyanine dyes,
listed in FIG. 5.
[0051] The synthetic steps in this example are as follows (using
standard Fmoc main-chain protection chemistry (Atherton &
Sheppard, "Solid Phase Peptide Synthesis: A Practical Approach",
IRL Press at Oxford University Press, Oxford, 1989, the contents
are included herein by reference)). TABLE-US-00005 TABLE V 1)
deprotect .alpha.-amino group 2) split resin population into a
small number of aliquots 3) for each resin aliquot, perform
sub-stoichiometric coupling with coding CyDye activated ester;
typical concentration: .apprxeq.0.001 to 0.1 mole of dye(s) per
mole of .alpha.-amino 4) for each resin aliquot, perform coupling
reaction with encoded amino acid 5) pool resin aliquots 6) repeat
steps 1-5 for each randomized position in the amino acid
sequence
[0052] This procedure avoids fluorescence energy transfer between
different dyes. First, labeling of any amino acid sequence as
described herein will inactivate and so will terminate that
sequence. Consequently, only a single dye is incorporated into any
sequence and intra-sequence energy transfer is avoided. Second, low
densities of dyes immobilized on the resin surface (see step 3
above) will ensure that lateral distances between labeled amino
acid sequences substantially exceed the pertinent Forster radii for
inter-strand fluorescent energy transfer. This is a manifestation
of the well known phenomenon of "pseudo-dilution" in solid phase
synthesis.
[0053] The practicability of the procedure in Table V has been
demonstrated by labeling standard combination synthesis bead resins
(NovaSyn TG amino resin, NovaBiochem, "Combinatorial Chemistry"
Catalog, San Diego, Calif., 1997, the contents of which are
included herein by reference). Specifically, we have constructed
SCC(l=1, m=6) as well as XSCC(l=1, m.sub.F=1, m.sub.I=5) with
individual dyes and with multiple dyes of the CyDye series and have
shown that colors are distinguishable by fluorescence microscopy at
molar ratios as low as 0.0001. In addition, we have demonstrated
that the dye coupling chemistry is compatible with protein
synthesis as specified in Table V.
[0054] The method of the present invention may be used to realize
color encoding of amino acid or peptide combinatorial libraries,
examples of which are summarized in Table VI. A suitable reporter
system is an anti-.beta.-endorphin monoclonal antibody (mAb)
directed against, an epitope in the form of an N-terminal amino
acid sequence N.sub.tes-YGGFL, where Y denotes tyrosine; binding of
the primary anti-.beta.-endorphin mAb to its target is detected by
a cascade-blue labeled secondary anti-mouse antibody (excitation at
396 nm, emission at 410 nm). TABLE-US-00006 TABLE VI Binary Color
Code (BCC) XXGFL-.beta.Ala-BEAD 16 = 4 .times. 4 species created
bit 1: Cy2 bit 3: Cy5 X = Gly, Ala, Tyr, Phe 16 = 2{circumflex over
( )}4 species created bit 2: Cy3 bit 4: Cy7 2-Level eXtended BCC
ZXXFL-.beta.Ala-BEAD 252 = 7 * 6 * 6 species created bit 1: Cy2 bit
5: Cy5 Z = Gly, Ala, Glu, Lys, 256 = 2{circumflex over ( )}8
species encoded bit 2: 2 * Cy2 bit 6: 2 * Cy5 Phe, Tyr, D-Tyr bit
3: Cy3 bit 7: Cy7 X = Gly, Ala, Glu, Lys, bit 4: 2 * Cy3 bit 8: 2 *
Cy7 Phe, Tyr 3-Level eXtended BCC XXXXL-.beta.Ala-BEAD 4096 =
8{circumflex over ( )}4 species created bit 1: Cy2 bit 7: Cy5 X =
Gly, Ala, Ser, Asn, 4096 = 2{circumflex over ( )}12 species encoded
bit 2: 2 * Cys2 bit 8: 2 * Cy5 Glu, Lys, Phe, Tyr bit 3: 4 * Cy2
bit 9: 4 * Cy5 bit 4: Cy3 bit 10: Cy7 bit 5: 2 * Cy3 bit 11: 2 *
Cy7 bit 6: 4 * Cy3 bit 12: 4 * Cy7
[0055] Although the method of the present invention is illustrated
by making reference to peptides and peptide precursors, the method
is equally suitable with any other chemical precursors and compound
classes that have been created via DCR combinatorial synthesis
(Calbiochem-NovaBiochem, "Solid Phase Organic Chemistry Handbook",
San Diego, Calif., 1997, the contents of which are included herein
by reference).
[0056] Compounds prepared by the disclosed methods have potential
use as therapeutic agents in the treatment of hypertension,
inflammation, and analgesia. For example, enkephalin analogues
selected by the disclosed methods may be useful as analgesics.
Organic compounds such as benzodiazepines useful as a muscle
relaxant may also be selected by the disclosed methods.
Diagnostics and Environmental Monitoring of Multiple Agents
[0057] The method of the present invention enables a novel
implementation of diagnostic assays and tests that probe
simultaneously for multiple reagents or pathogens. In contrast to
the spatial encoding of diagnostic panels in all prior art, random
assemblies of multiple bead types, distinguishable by their
respective color codes, can be mixed and handled in parallel. For
example, the implementation of bead-based immunodiagnostic assay
formats can take advantage of color coding as described herein to
display a multiplicity of specific bead-anchored antibodies, each
type assigned to a specific color code, to monitor for a
multiplicity of agents in the ambient.
[0058] A preferred implementation of a multi-agent diagnostic assay
uses random arrays of chemically encoded beads (FIG. 6). For
example, the determination of blood type would require only five
distinct bead types, a task that is readily addressed by the SCC
(l=1, m=5). This realization of diagnostic testing and
environmental monitoring devices would facilitate miniaturization,
integration of multiple tests and automated operation relying on
spectral read-out.
In-Situ Interrogation and Decoding of Color-Encoded Beads
[0059] The optical arrangement in FIG. 7 provides for the
integration of two essential capabilities: fluorescence microscopic
imaging and multi-color fluorescence analysis of individual beads.
The latter serves to determine the relative abundances of several
fluorophores present on the bead surface.
[0060] The use of a microscope objective of high numerical aperture
(N.A. =0.7)(702) serves to maximize collection efficiency as well
as spatial resolution. The principal additional components of FIG.
7 are: a long-pass filter to reject stray excitation light (704), a
dichroic beam splitter (706) to separate beams for image formation
by the field lens (708) and spectral analysis via focusing of the
light (by lens 710) on the slit aperture of a grating monochromator
(712) or, alternatively (not shown), on the entrance pupil of an
optical fiber that is coupled to a grating monochromator;
multi-color spectra are recorded by a CCD array (714).
Infinity-corrected optical components offer convenience of
implementation.
[0061] While simple long pass filters have been employed in DNA
sequencing applications to reject stray excitation light supplied
at a single wavelength, interference filters can be designed to
provide multiple narrow (10 nm) pass-bands at several emission
wavelengths characteristic of the CyDye family of fluorophores
discussed herein. Similar fabrication techniques may be applied to
the dichroic mirror. These considerations are particularly relevant
to an epi-fluorescence geometry, a special case of reflection
microscopy.
[0062] Among the suitable instrumental realizations of recording
spectral information from individual color-encoded beads or
collections of color-encoded beads are flow cytometric analysis and
multi-spectral imaging. The latter permits the collection of
spectral information from individual or multiple beads in the field
of view of a microscope or other imaging device, as considered in
FIG. 7.
[0063] Methods suitable for multi-spectral imaging include:
multiplexing of distinct wavelengths of incident and emitted light
and illumination with a superposition of multiple wavelengths,
followed by dispersive imaging by means of a grating or prism (see
FIG. 7) or followed by interferometric analysis of emitted
light.
[0064] The first method is readily implemented using matching
optical pass-band filters; these are mounted in filterwheels and
positioned in incident and emitted light paths of a microscope. The
synchronized rotation of the two filterwheels will insert matching
pairs of excitation and emission filters (a reflective geometry
will also require a suitable dichroic mirror) into the light path,
producing a repeating series of images at each of the distinct
wavelengths selected one of the filter/mirror combination. This
principle is realized, for example in the Fluorescence Imaging
MicroSpectrophotometer developed by Kairos Scientific (Santa Clara,
Calif.).
[0065] In the second method, distinct wavelengths for illumination
are produced by a multi-pass band filter/mirror combination; a
prism is inserted into the output path. This configuration
facilitates the imultaneous spectral analysis of multiple beads
located in a rectangular slice of the field of view of the
microscope. Light emitted from beads within this slice is imaged
onto the entrance slit of the prism and is decomposed into its
spectral components. This principle is realized in the PARISS
Imaging Spectrometer attachment developed by LightForm (Belle
Meade, N.J.). In the third method, light from the entire field of
view is analyzed inteferometrically: a pellicle beamsplitter in the
output path produces two (coherent) light beams which are reflected
by a mirror and recombined. As the beamsplitter is rotated, a small
difference in pathlength is introduced between the two light beams,
resulting in interference fringes as the two beams are recombined.
These fringes contain the entire spectral information contained in
the light emiited from the field of view of a microscope (Garini et
al, Bioimaging 4, 65-72 (1996)). That is, as the beamsplitter is
rotated, a continuous spetrum is generated for every position
within the field of view, resulting in a three-dimensional
representation of the data. This principle is realized in the
SpectraCube system developed and marketed by Applied Spectral
Imaging (Carlsbad, Calif.). In contrast to the first method, the
second and third methods generate a continuous spectrum,
facilitating spectral classification of overlapping emission
bands.
[0066] The arrangements in FIG. 8 provide for additional
flexibility in rejecting stray light by spatially separating
incident light and emitted light collection in transmission and
rejection microscopy, as illustrated in FIGS. 8A and 8B,
respectively. In addition, the use of specially deigned multi-pass
band interference filters in the output light path is again an
option.
[0067] The demands on the sensitivity of the multi-color
fluorescence detection system derive from the number of
fluorophores of each color expected to be present on a selected
bead. A bead of radius R and surface area A=4.pi.R 2 will
accommodate up to N=A/a molecules of molecular area a, or N{dot
over ( )}=xN fluorophores. With a=30A and 0.01<x<0.1, a bead
of 10 .mu.m diameter may carry 10 7.ltoreq.N{dot over ( )}10 8
flurophores. For comparison, imaging of small circular domains of
10 .mu.m diameter within a monomolecular film composed of a
phospholipid containing 1 mole % of a fluorescent analog and
confined to an air-water interface, is based on a comparable number
of fluorophores and is readily accomplished using
silicon-intensified target (SIT) camera technology. The refractive
property of beads in aqueous solution will fuirher enhance the
light collection efficiency of the entire system.
In-Situ Interrogation and Decoding of Color-Encoded Bead Arrays
[0068] The present invention provides a methodology for
color-encoding of beads and describes a method and apparatus for
in-situ interrogation and decoding of color-encoded beads and
collections of beads by multi-color fluorescence imaging and
spectral analysis. This method is compatible with all bead assay
formats described to date, as discussed herein.
[0069] A preferred format providing a particularly efficient
realization of bead assays on the basis of the methods and
apparatus of the present invention involves planar beads arrays.
This format facilitates highly parallel screening of enzyme
activity, receptor-ligand binding, antibody-antigen recognition as
well as DNA or RNA hybridization, etc. Thus, a close-packed array
of 100 .mu.m diameter beads can contain of the order of 10 4 beads
in an area of only 1 cm 2, permitting the examination of up to 10 4
compounds/cm 2 in a single pass. The instantaneous determination of
chemical identities enables the efficient implementation of
re-iterative screening in which multiple copies of each bead type
are examined to establish a statistically robust ranking of
compounds producing positive assay scores. Furthermore, the
implementation of the present invention in a planar bead array
format lends itself to automation. Automated operation would entail
the preparation of planar bead arrays, followed by fluorescence
imaging of the array to locate beads that are to be subjected to
spectral analysis and on-line decoding. The intrinsic detection
sensitivity of fluorescence, demonstrated at the level of detecting
single fluorophores, makes it possible to substantially reduce the
size of synthesis beads. This in turn facilitates miniaturization
and containment within an enclosed system, with its attendant
benefits of reducing the requisite quantity of synthesized compound
and the amount of reagents consumed in tie course of screening.
[0070] One method of forming planar bead arrays is to rely on
gravity-driven settling of beads from suspension to produce a
(static) layer of beads or arrangement of bead clusters on a planar
substrate. A second method employs dynamic planar bead arrays that
are formed adjacent to planar surfaces and manipulated in-situ
under external control, for example by Light-controlled
Electrokinetic Assembly of Particles near Surfaces (LEAPS). LEAPS
is a technology that provides the capability to form dynamic planar
bead arrays in aqueous solution on cue and to place and maintain
them in a designated area of a planar electrode surface, as set
forth in the copending PCT application filed Apr. 24, 1997,
entitled "Light Controlled Electrokinetic Assembly of Particles
Near Surfaces", based on U.S. Provisional Application Ser. No.
60/016,642, filed Apr. 25, 1996, which is incorporated by reference
herein.
[0071] Dynamic planar bead arrays provide additional advantages in
the realization of automated screening assays in a miniaturized,
contained environment. Bead suspensions from a synthesis pool will
be loaded into a "sandwich" flow cell where planar bead arrays are
formed adjacent to the planar walls of cell; screening assays will
be performed in planar array format to identify lead compounds
without the need of a time-consuming and error-prone step of
physical separation; following completion of the scheduled assays,
bead arrays will be dis-assembled and the bead suspension
discharged to ready the flow cell for another cycle. In the
example, a redundancy of 10, i.e., the presence of 10 copies of
beads of identical type and color code, would still facilitate
screening of 1000 compounds at a time, but would considerably
enhance the quality of any pharmacokinetic characterization. The
benefits of miniaturization would be enhanced by the use of small
synthesis beads. Chemically and physically well defined beads in
the requisite size range (10 .mu.m diameter) are available from
many commercial sources. They are readily manipulated by LEAPS to
form dynamic planar bead arrays of high density. This ensures that
screening assays may be performed in a highly parallel format on a
large number of samples, and this in turn provides the basis for
highly re-iterative screening and for a robust pharmacokinetic
characterization of potential lead compounds.
[0072] The present invention will be better understood from the
Experimental Details which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described in
the claims which follow thereafter.
EXAMPLE 1
1. Color-Encoded Peg-Polystyrene Microspheres
a. Preparation of Color-Encoded Peg-Polystyrene Microspheres
[0073] (1) Cy2 (ex=489 nm, em=506 nm)-color-encoded PEG-polystyrene
microspheres: 50 mg of NovaSyn TG anmino microspheres (NovaBiochem;
130.mu. diameter, 15 .mu.mol amine) were equilibrated in 10 ml DMF
30 min at 25.degree. C. The supernatant was removed by filtration,
and 100 .mu.l DMF, 1 .mu.l TEA and 15 .mu.l 1 mM Cy2-bisfunctional
NHS-ester (Amersham; 15 nmol) were added in DMF. The reaction
mixture was shaken 1 h at 25.degree. C., 2 .mu.l (20 .mu.mole)
n-butylamine was added, and the reaction mixture was shaken a
further 30 min at 25.degree. C. The supernatant was removed, and
microspheres were washed twice with 5 ml DMF, rinsed twice with 5
ml chloroform and dried in vacuo.
[0074] (2) Cy3 (ex=550 nm, em=570 nm)-color-encoded PEG-polystyrene
microspheres: This preparation was identical to (1) except that, in
parallel reactions, 15 .mu.l of 0.001, 0.01, 0.1, and 1 mM
Cy3-monofunctional NHS-ester (Amersham; 0.15, 1.5, and 15 nmol)
were used, and the n-butylamine step was omitted.
(3) Cy3.5 (ex=581 nm, em=596 nm)-color-encoded PEG-polystyrene
microspheres: This preparation was identical to (1) except that 15
.mu.l of 1 mM Cy3.5-monofunctional NHS-ester (Amersham; 15 nmol)
was used, and the n-butylamine was step omitted.
(4) Cy5 (ex=649 nm, em=670 nm)-color-encoded PEG-polystyrene
microspheres: This preparation was identical to (1) except that 15
ul of 1 mM Cy5-monofunctional NHS-ester (Amersham; 15 nmol) was
used, and the n-butylamine step was omitted.
(5) Cy5.5 (ex=675 nm, em=694 nm)-color-encoded PEG-polystyrene
microspheres: This preparation was identical to (1) except that 15
ul of 1 mM Cy5.5-monofunctional NHS-ester (Amersham; 15 nmol) was
used, and the n-butylamine step was omitted.
(6) Cy7 (ex=743 nm, em=767 nm)-color-encoded PEG-polystyrene
microspheres: This preparation was identical to (1) except that 15
.mu.l of 1 mM Cy7-bisfunctional NHS-ester (Amersham; 15 nmol) was
used.
(7) Cy3/Cy5-color-encoded PEG-polystyrene microspheres: This
preparation was identical to (1) except that both
Cy3-monofunctional NHS-ester and Cy5-monfunctional NHS-ester were
added (15 .mu.l of 1 mM stock each), and the n-butylamine step was
omitted.
(8) Cy2/Cy3/Cy5/Cy7-color-encoded PEG-polystyrene microspheres:
This preparation was identical to (1) except that Cy2-bisfunctional
NHS-ester, Cy3-monofunctional NHS-ester, Cy5-monofunctional
NHS-ester, and Cy7-bisfunctional NHS-ester were added (15;l of 1 mM
stock each).
b. Stability of Cy3-Encoded Peg-Polystyrene Microspheres to
Solid-Phase Peptide Synthesis Conditions.
[0075] Cy3-encoded PEG-polystyrene microspheres were subjected to
one cycle of solid-phase peptide synthesis. 50 mg microspheres and
5 mg Fmoc(Lys)Boc-OBT [prepared by reacting 94 mg Fmoc(Lys)Boc-OH
(NovaBiochem; 0.2 mmol), 48 mg DCC (Aldrich; 0.22 mmol) and 27 mg
HOBT (Aldrich; 0.2 mmol) in 2 ml DMF for 0.5 h at 25.degree. C.,
centrifuging at 2000.times. g 5 min at 25.degree. C., and using 100
.mu.l of the supernatant) in 100 .mu.l DMF were shaken 0.5 h at
25.degree. C. The microspheres were filtered, suspended in 100
.mu.l 20% piperidine in DMF 15 min at 25.degree. C., washed twice
with 5 ml CHCl.sub.3, and dried. The UVNIS absorbance and
fluoresence properties of the Cy3-encoded PEG-polystyrene
microspheres were unchanged.
c. Optical Properties of Color-Encoded Peg-Polystyrene
Microspheres
Microspheres examined for their optical properties included:
[0076] Cy3 (ex=550 nm, em=570 nm)-color-encoded PEG-polystyrene
microspheres of four different intensity levels, prepared as
described in section a-(2) above by reacting beads with 0.001,
0.01, 0.1 and 1 mM Cy3, are denoted b3-0001, b3-001, b3-01 and
b3-1, respectively; as a group, all the Cy3-encoded PEG-polystyrene
microspheres are denoted b3-x. Cy5 (ex=649 nm, em=670
nm)-color-encoded PEG-polystyrene microspheres, prepared as
described in section a-(2) above by reacting beads with 1 mM Cy5,
are denoted b5-1; Cy3/Cy5-color-encoded PEG-polystyrene
microspheres, prepared as described in section a2) above by
reacting beads with 1 mM Cy3/Cy5, are denoted b35-1.
An aliqout of dried microspheres was suspended in DMF and dispersed
on a silicon wafer; DMF was evaporated by gentle heating. All
subsequent observations were made in air.
(1) Fluorescence Imaging
[0077] Observations were made with a Zeiss UEM microscope equipped
for epifluorescence; combinations of excitationfilter/dichroic
mirror/emission filter designed for Cy3 and Cy5 (Chroma
Technologies, Brattleboro, Vt.) were used in conjunction with a
100W halogen illuminator and objectives of 10.times., 25.times. and
40.times. magnification. Optionally, images were recorded with a
SIT camera (Cohu, San Diego, Calif.).
[0078] All microspheres displayed a bright circumferential "ring"
of high intensity, corresponding to .ltoreq.5% of the particle
diameter, suggesting that label was associated primarily with the
surface, rather than the interior, of each particle. Even the
dimmest particles, of type b3-0001, were readily observable using a
25.times./0.45 NA objective and the SIT camera.
[0079] Microspheres of type b3-0001 appeared dimmer than did
microspheres of type b3-001, although by less than the expected
factor of 10. This phenomenon remains to be explored, but may
indicate fluorescence quenching. Any given set of Cy3-encoded
microspheres displayed particle-to-particle variations in color:
some particles appeared orange, others yellow of type b5-1 appeared
bright red.
(2) Fluorescence Spectra
[0080] To demonstrate the feasibility of in-situ interrogation of
color-encoded microspheres, fluorescence spectra were recorded from
individual color-encoded PEG-polystyrene microspheres by means of a
PARISS.TM. imaging spectrophoto-meter (prototype supplied by
LightForm, Belle Meade, N.J.) with 50 .mu.m wide entrance slit,
curved prism and room-temperature CCD array capable of on-chip
integration. The instrument was mounted to the camera port of a
Zeiss UEM microscope. In this configuration, multiple beads which
are lined up along the long dimension of the projected slit can be
imaged and spectrally analyzed. Only an approximate wavelength
calibration was performed.
[0081] Spectra displaying fluorescence intensity as a function of
wavelength were obtained separately for Cy3- and for Cy5-encoded
microspheres and showed the following spectral characteristics:
[0082] b3-x: spectra were obtained for all types of particles;
specific features included: for b3-0001: signal-to-noise
(S/N).varies.2, signal-to-background (S/B).varies.1.5; for b3-001:
S/N 4, S/B.varies.2 (with a CCD integration time of approximately
10 s); smoothing clearly revealed characteristic spectral features;
for b3-1: SN>10;
b5-1: very clean spectra were recorded, all with a slight skew
toward high wavelength;
[0083] b35-1: very clean spectra of either label were recorded,
switching between appropriate filters to simulate filter wheel
operation. At this concentration, spectra (taken with 10-times
shorter integration time than that used for b3-01 and b3-001)
displayed no discernible noise.
2. Color-Encoded Macroporous Polystyrene Microspheres
a. Preparation of Color-Encoded Macroporous Polystyrene Micro
Spheres
[0084] 50 mg Amino-Biolinker-PMI-1000 amino oligoethylene
glycol-functionalized macroporous polystyrene microspheres (Solid
Phase Sciences; 35.mu. diameter, 7 .mu.mol amine) were equilibrated
in 2 ml DMF 20 min at 25.degree. C. The supernatant was removed by
filtration, and 100 .mu.l DMF, 1 .mu.l TEA, and 70 .mu.l 1 mM
Cy3-monofunctional NHS-ester (Amersham; 70 nmol) were added. After
1 hr at 25.degree. C. with shaking, the supernatant was removed by
filtration, and the microspheres were washed twice with 5 ml DMF,
washed twice with 5 ml CHCl.sub.3, and dried in vacuo.
b. Optical Properties of Color-Encoded Macroporous Polystyrene
Microspheres
[0085] Visual inspection using the configuration descibed under
Example 1, revealed substantial bead-to-bead variations in
fluorescence intensity.
3. Color-Encoded Solid Glass Microspheres ("Pelicular
Microspheres")
a. Preparation of Color-Encoded Pelicular Microspheres
(1) Epoxide-Functionalized Pelicular Microspheres:
[0086] 4 g solid sodalime glass microspheres (Duke Scientific;
40+31 diameter; 4.8.times.10' microspheres), 7 ml xylene, 2.34 ml
3-glycidoxypropyltrimethoxysilane (Aldrich; 1 mmol) and 0.117 ml
diisopropylethylamine (Aldrich; 0.7 mmol) were shaken 18 h at
80.degree. C. Upon cooling to room temperature, microspheres were
filtered, washed with 40 ml methanol, washed with 40 ml diethyl
ether, and dried in vacuo.
(2) MMT-NH-PEG-Functionalized Pelicular Microspheres:
[0087] Microspheres from (1) were suspended in a solution of 200 mg
mono-MMT-1,13-trioxotridecadiamine [0.4 mmol; prepared by mixing 7
g MMT-Cl (Aldrich; 23 mmol) and 11.3 ml
4,7,10-trioxa-1,13-tridecanediamine (Aldrich; 51 mmol) in 150 ml
1:1:1 methylene chloride:pyridine:acetonitrile for 18 h at
25.degree. C., then isolating the required adduct by chromatography
on silica gel) in 6 ml xylene. Approximately 10 mg sodium hydride
(Aldrich; 0.4 mmol) was added, and the suspension shaken 18 h at
40.degree. C. under a drying tube. Microspheres then were filtered
and successively washed with 20 ml methanol, 10 ml water, ml
methanol, and 20 ml chloroform, and dried in vacuo.
[0088] Dried microspheres were capped by reaction with 5% acetic
anhydride, 5% 2,6-lutidine, 8% N-methylimidazole in 10 ml
tetrahydrofirmn 1 h at 25.degree. C. with shaking, successively
washed in 2.times.5 ml methanol, 2.times.5 ml chloroform, and
2.times.5 ml diethyl ether, and dried in vacuo.
(3) H.sub.2N-PEG-Functionalized Pelicular Microspheres:
Microspheres from (2) were treated with 1 ml 3% TFA in
CH.sub.2Cl.sub.20.5 h at 25.degree. C. with shaking. Based on
quantitation of released monomethoxy trityl cation
(.epsilon..sub.478=3.47.times.10.sup.4 M.sup.-1 cm.sup.-1) the
loading densities of H.sub.2N-PEG were as follows:
[0089] 15 fmol H.sub.2N-PEG per microsphere [0090]
1.1.times.10.sup.10 molecules H.sub.2N-PEG per microsphere [0091]
0.022 molecule H.sub.2N-PEG per .ANG..sup.2 Assuming .apprxeq.0.04
available silanol groups per .ANG..sup.2 of soda-lime glass, the
grafting efficiency was .apprxeq.50%. (4) Color-Encoded
Peg-Functionalized Pelicular Microspheres: To 20 mg of
H.sub.2N-PEG-functionalized pelicular microspheres (4.2 nmol
amine), were added 97 .mu.l DMF, 2 .mu.l TEA, and 0.8 .mu.l 1 mM
Cy3-monofunctional NHS-ester (Amersham; 0.8 nmol), and the
resulting suspension was shaken for 18 h at 25.degree. C.
Microspheres then were filtered and washed successively with 5 ml
DMF, 5 ml methanol, 5 ml chloroform, and 5 ml diethyl ether, and
dried in vacuo. Based on quantitation of consumed
Cy3-monofunctional NHS-ester (.epsilon..sub.552=1.5.times.10.sup.5
M.sup.-1 cm.sup.-1) the loading of Cy3 densities were as follows:
[0092] 1 frnol Cy3 per microsphere [0093] 6.times.10.sup.8
molecules Cy3 per microsphere [0094] 0.001 molecule Cy3 per
.ANG..sup.2 [0095] 0.07 molecule Cy3 per molecule available
H.sub.2N-PEG b. Optical Properties of Cy3-Encoded
Peg-Functionalized Pelicular Microspheres: Visual inspection using
the configuration described under Example 1, revealed uniformly
fluorescent microspheres.
* * * * *