U.S. patent application number 09/047119 was filed with the patent office on 2001-07-05 for small molecule library screening using facs.
Invention is credited to PAYAN, DONALD.
Application Number | 20010006787 09/047119 |
Document ID | / |
Family ID | 21947163 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006787 |
Kind Code |
A1 |
PAYAN, DONALD |
July 5, 2001 |
SMALL MOLECULE LIBRARY SCREENING USING FACS
Abstract
The present invention relates to methods of screening libraries
of small molecules such as combinatorial chemical libraries of
organic molecules, including peptides and other chemical libraries,
for binding to target molecules, using fluoroscence-activated cell
sorting (FACS) machines.
Inventors: |
PAYAN, DONALD;
(HILLSBOROUGH, CA) |
Correspondence
Address: |
RICHARD F. TRECARTIN
FLEHR HOHBACH TEST ALBRITTON & HERBERT
FOUR EMBARCADERO CENTER
SUITE 3400
SAN FRANCISCO
CA
941114187
|
Family ID: |
21947163 |
Appl. No.: |
09/047119 |
Filed: |
March 24, 1998 |
Current U.S.
Class: |
435/7.1 ;
435/7.2; 435/7.21; 435/7.23; 435/7.5; 435/7.9; 435/7.92; 435/7.94;
436/172; 530/387.1 |
Current CPC
Class: |
C40B 30/04 20130101;
G01N 33/585 20130101; G01N 33/6845 20130101; G01N 33/54313
20130101; B01J 2219/00702 20130101 |
Class at
Publication: |
435/7.1 ;
436/518; 435/7.2; 435/7.21; 435/7.23; 435/7.5; 435/7.9; 435/7.92;
435/7.94; 436/172; 530/387.1 |
International
Class: |
G01N 033/53; G01N
033/543; C07K 016/00; G01N 021/76 |
Claims
I claim:
1. A method for screening candidate bioactive agents for binding to
a target molecule comprising a) contacting a library of candidate
bioactive agents covalently attached to a plurality of beads with
at least a first population of first target molecules labeled with
a first labeling moiety; b) adding a fluorescent second labeling
moiety capable of binding to said first labeling moiety; and c)
sorting the beads using a fluorescent activated cell sorter (FACS)
machine to obtain a population of fluorescent beads and a
population of non-fluorescent beads; wherein the presence of at
least one fluorescent bead is indicative that at least one
candidate bioactive agent that binds to at least one target
molecule.
2. A method for screening candidate bioactive agents for binding to
a target molecule comprising a) contacting a library of candidate
bioactive agents covalently attached to a plurality of beads with
at least a first population of first target molecules comprising at
least a first subpopulation labeled with a first labeling moiety
and a second subpopulation labeled with a second labeling moiety;
b) optionally adding a third labeling moiety capable of binding to
said first labeling moiety, wherein at least one of said first and
said third labeling moieties is fluorescent; c) optionally adding a
fourth labeling moiety capable of binding to said second labeling
moiety, wherein at least one of said second and said fourth
labeling moieties is fluorescent; and d) sorting the beads using a
fluorescent activated cell sorter (FACS) machine to obtain a
population of fluorescent beads and a population of non-fluorescent
beads; wherein the presence of at least one fluorescent bead is
indicative that at least one candidate bioactive agent binds to at
least one first target molecule.
3. A method according to claim 2 wherein said third labeling moiety
is added.
4. A method according to claim 2 or 3 wherein said fourth labeling
moiety is added.
5. A method according to claim 1 or 2 wherein said beads are added
to a second population of second target molecules, wherein said
second target molecules are labelled with at least a different
fluorescent label than the fluorescent label(s) of said first
population of first target molecules.
6. A method for screening candidate bioactive agents for binding to
a target molecule comprising: a) contacting a library of candidate
bioactive agents covalently attached to a plurality of beads with
at least a first population of first target molecules labeled with
a first labeling moiety; b) optionally adding a second labeling
moiety capable of binding to said first labeling moiety, wherein at
least one of said first and said second labeling moieties is
fluorescent; c) sorting the beads using a fluorescent activated
cell sorter (FACS) machine to obtain a population of fluorescent
beads and a population of non-fluorescent beads; d) adding to said
fluorescent beads a competitor moiety known to bind to said first
target; e) resorting the beads by a FACS machine to produce a
population of beads which are no longer fluorescent; wherein the
presence of at least one bead which was fluorescent in step (c) but
is no longer fluorescent in step (e) indicates that at least one
candidate bioactive agent is a bioactive agent that binds to said
first target molecule.
7. A method according to claim 6 wherein said competitor moiety is
an antibody.
8. A method according to claim 1, 2 or 6 further comprising
altering an experimental reaction condition and resorting said
fluorescent beads.
9. A method according to claim 8 wherein said experimental reaction
condition is temperature, salt concentration or pH.
10. A method according to claim 1, 2 or 6 further comprising
characterizing the binding agent which binds to said target
molecule.
11. A method according to claim 1, 2 or 6 further comprising
synthesizing said candidate bioactive agents on said beads.
12. A method for screening candidate bioactive agents for binding
to a target molecule comprising: a) contacting a library of
candidate bioactive agents covalently attached to a plurality of
beads to at least a first population of first target molecules
labeled with a first labeling moiety; b) optionally adding a second
labeling moiety which will bind to said first labeling moiety,
wherein at least one of said first and said second labeling
moieties is fluorescent; c) sorting the beads using a fluorescent
activated cell sorter (FACS) machine to obtain a population of
fluorescent beads and a population of non-fluorescent beads; d)
chemically modifying said candidate bioactive agents of said
population of non-fluorescent beads to form new candidate bioactive
agents.
13. A method according to claim 12 further comprising: e) adding
the beads containing the new candidate bioactive agents with said
first population of first target molecules; and f) resorting the
beads by a FACS machine.
14. A method for screening candidate bioactive agents for binding
to a target molecule comprising: a) contacting a library of
candidate bioactive agents covalently attached to a plurality of
beads to at least a first population of first target molecules
labeled with a first labeling moiety; b) optionally adding a second
labeling moiety which will bind to said first labeling moiety,
wherein at least one of said first and said second labeling
moieties is fluorescent; c) sorting the beads using a fluorescent
activated cell sorter (FACS) machine to obtain a population of
fluorescent beads and a population of non-fluorescent beads; d)
removing the bound target molecules from said fluorescent beads to
form a population of intermediate beads; e) chemically modifying
said candidate bioactive agents of said population of intermediate
beads to form new candidate bioactive agents.
15. A method according to claim 14 further comprising: e) adding
the beads containing the new candidate bioactive agents with said
first population of first target molecules; and f) resorting the
beads by a FACS machine.
16. A method according to claim 1, 2, 6, 12 or 14 further
comprising testing the bioactive agent which binds to said target
molecule in a cell containing said target molecule.
17. A method according to claim 16 further comprising testing the
bioactive agent which binds to said target molecule in a cell
containing said target molecule.
18. A method according to claim 1, 2, 6, 12 or 14 further
comprising removing any bound target molecules from said beads and
retesting the beads on a second population of second target
molecules.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of screening
libraries of small molecules such as combinatorial chemical
libraries of organic molecules, including peptides and other
chemical libraries, for binding to target molecules, using
fluoroscence-activated cell sorting (FACS) machines.
BACKGROUND OF THE INVENTION
[0002] Traditional approaches to identify and characterize new and
useful drug candidates include the isolation of natural products or
synthetic preparation, followed by testing against either known or
unknown targets. See for example WO 94/24314, Gallop et al., J.
Med. Chem. 37(9):1233 (1994); Gallop et al., J. Med. Chem.
37(10):1385 (1994); Ellman, Acc. Chem. Res. 29:132 (1996); Gordon
et al., E. J. Med. Chem. 30:388s (1994); Gordon et al., Acc. Chem.
Res. 29:144 (1996); WO 95/12608, all of which are incorporated by
reference.
[0003] The screening of these libraries is done in a variety of
ways. One approach involves attachment to beads and visualization
with dyes; see Neslter et al., Bioorg. Med. Chem. Lett. 6(12): 1327
(1996). Another approach has utilized beads and fluorescence
activated cell sorting (FACS); see Needles et al., PNAS USA
90:10700 (1993), and Vetter et al., Bioconjugate Chem. 6:319
(1995).
[0004] Fluorescence activated cell sorting (FACS), also called flow
cytometry, is used to sort individual cells on the basis of optical
properties, including fluorescence. It is generally fast, and can
result in screening large populations of cells in a relatively
short period of time.
[0005] Accordingly, it is an object of the invention to provide
methods for the rapid, accurate screening of candidate agents,
particularly libraries of agents, using FACS methods.
SUMMARY OF THE INVENTION
[0006] In accordance with the objects outlined above, the present
invention provides methods for screening candidate bioactive agents
for binding to a target molecule. The methods comprise contacting a
library of candidate bioactive agents covalently attached to a
plurality of beads with at least a first population of first target
molecules labeled with a first labeling moiety. A fluorescent
second labeling moiety capable of binding to said first labeling
moiety is then added, and the beads are sorted using a fluorescent
activated cell sorter (FACS) machine to obtain a population of
fluorescent beads and a population of non-fluorescent beads. The
presence of at least one fluorescent bead is indicative that at
least one candidate bioactive agent that binds to at least one
target molecule.
[0007] In an additional aspect, the methods comprise contacting a
library of candidate bioactive agents covalently attached to a
plurality of beads with at least a first population of first target
molecules comprising at least a first subpopulation and a second
subpopulation. The first subpopulation is labeled with a first
labeling moiety (and optionally with a third labeling moiety) and
the second subpopulation is labeled with a second labeling moiety
(and optionally with a fourth labelling moiety). The beads are then
sorted using a fluorescent activated cell sorter (FACS) machine to
obtain a population of fluorescent beads and a population of
non-fluorescent beads, wherein the presence of at least one
fluorescent bead is indicative that at least one candidate
bioactive agent binds to at least one first target molecule.
[0008] In a further aspect, the methods comprise contacting a
library of candidate bioactive agents covalently attached to a
plurality of beads with at least a first population of first target
molecules labeled with a first labeling moiety (and optionally a
second labeling moiety), and sorting the beads using a fluorescent
activated cell sorter (FACS) machine to obtain a population of
fluorescent beads and a population of non-fluorescent beads. A
competitor moiety known to bind to the first target is then added,
and the beads are resorted by a FACS machine to produce a
population of beads which are no longer fluorescent, wherein the
presence of at least one bead which was fluorescent in step (c) but
is no longer fluorescent in step (e) indicates that at least one
candidate bioactive agent is a bioactive agent that binds to said
first target molecule.
[0009] In an additional aspect, the methods of the invention
further comprise chemically modifying the candidate bioactive
agents to form new candidate bioactive agents, which may then be
rescreened using the methods of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The present invention relates to methods of screening
candidate agents for the capacity to bind to a target molecule.
Generally speaking, a preferred embodiment of the invention is
described as follows. Many synthetic reactions for small molecules
(organic moieties, peptides, nucleic acids, etc.) are amenable to
solid phase synthesis. Thus, solid phase synthesis may be done on
support particles or beads that can be sorted by FACS machines. In
this way, a library of beads containing a library of candidate
bioactive agents can be made. The number of candidate bioactive
agents can vary, as will the number of beads which contain a
particular candidate agent.
[0011] Once generated, the library of beads is added to a
population of target molecules for which an interaction is sought.
The target molecules are labelled with a first labeling moiety,
which can either be a fluorescent tag or a molecule to which a
second label which is fluorescent can be added. Thus for example,
the target molecule may be labeled either directly with a
fluorescent tag, or with a hapten such as biotin, followed by
treatment with a a fluorescently labeled second moiety such as
streptavidin (or both). The latter technique may be particularly
advantageous to "amplify" the fluorogenicity of the target, thus
allowing smaller amounts of target to be used and/or detected. In
either case the target molecule is ultimately fluorescent. The
beads are added, and allowed to interact under conditions which
will favor binding of a candidate bioactive agent and a target
molecule. The beads are then subjected to sorting by FACS, which
allows the beads with bound fluorescent targets, and thus bioactive
agents, to be separated from the beads which do not contain
fluorescent targets.
[0012] Optionally, the beads containing the fluorescent targets may
be further tested by adding a binding moiety known to bind to the
target molecule, such as an antibody, peptide, binding partner,
ligand, etc. Under certain circumstances, there may be competitive
binding as between the bioactive agent and the binding moiety, with
the binding moiety displacing the bioactive agent. The beads can
then be resorted, and those beads which were fluorescent prior to
the addition of the binding moiety but are now non-fluorescent due
to the displacement of the fluorescent bioactive agent by a
non-fluorescent binding partner can be collected and analyzed.
[0013] The same techniques can be used to screen multiple targets
simultaneously, with each target containing a different
fluorophore. Alternatively, a population of target molecules can be
separated into subpopulations, with the same target molecule of
each subpopulation being labelled with a different fluorophore, in
order to reduce the signal for non-specific binding, i.e. only
beads which contain more than one fluorophore are selected.
[0014] Once identified, the beads containing the fluorescent
targets can then be treated in a variety of ways. The targets can
be removed and the bioactive agent which bound to the target can be
characterized structurally and biochemically, and tested in vivo,
if desired, or subjected to more rigorous biochemical and
functional screens. The bioactive agent may be further modified to
increase affinity or specificity, elucidate functional mechanisms
or binding areas, etc.
[0015] In addition, beads which did not bind targets (as well as
beads that did bind targets) can be subjected to further solid
phase synthesis. That is, the present methods allow the screening
of combinatorial libraries as they are synthesized, at the end of
each step, thus eliminating the requirement for a defined "end
point" of synthesis.
[0016] Furthermore, a distinct advantage of the present invention
is that the beads containing the libraries are reusable, and may be
screened against any number of targets without requiring new
synthesis of the candidate compounds.
[0017] Accordingly, the present invention provides methods for
screening candidate bioactive agents for binding to a target
molecule. By "candidate bioactive agent" or "candidate drugs" or
grammatical equivalents herein is meant any molecule, e.g. proteins
(which herein includes proteins, polypeptides, and peptides), small
organic or inorganic molecules, polysaccharides, polynucleotides,
etc. which are to be tested against a particular target. Candidate
agents encompass numerous chemical classes. In a preferred
embodiment, the candidate agents are organic molecules,
particularly small organic molecules, comprising functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more chemical
functional groups.
[0018] Candidate agents are obtained from a wide variety of
sources, as will be appreciated by those in the art, including
libraries of synthetic or natural compounds. As will be appreciated
by those in the art, the present invention provides a rapid and
easy method for screening any library of candidate agents,
including the wide variety of known combinatorial chemistry-type
libraries.
[0019] In a preferred embodiment, candidate agents are synthetic
compounds. Any number of techniques are available for the random
and directed synthesis of a wide variety of organic compounds and
biomolecules, including expression of randomized oligonucleotides.
See for example WO 94/24314, hereby expressly incorporated by
reference, which discusses methods for generating new compounds,
including random chemistry methods as well as enzymatic methods. As
described in WO 94/24314, one of the advantages of the present
method is that it is not necessary to characterize the candidate
bioactive agents prior to the assay; only candidate agents that
bind to the target need be identified. In addition, as is known in
the art, coding tags using split synthesis reactions may be done,
to essentially identify the chemical moieties on the beads.
[0020] Alternatively, a preferred embodiment utilizes libraries of
natural compounds in the form of bacterial, fungal, plant and
animal extracts that are available or readily produced, and can be
attached to beads as is generally known in the art.
[0021] Additionally, natural or synthetically produced libraries
and compounds are readily modified through conventional chemical,
physical and biochemical means. Known pharmacological agents may be
subjected to directed or random chemical modifications, including
enzymatic modifications, to produce structural analogs.
[0022] In a preferred embodiment, candidate bioactive agents
include proteins, nucleic acids, and chemical moieties.
[0023] In a preferred embodiment, the candidate bioactive agents
are proteins. By "protein" herein is meant at least two covalently
attached amino acids, which includes proteins, polypeptides,
oligopeptides and peptides. The protein may be made up of naturally
occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures. Thus "amino acid", or "peptide residue",
as used herein means both naturally occurring and synthetic amino
acids. For example, homo-phenylalanine, citrulline and noreleucine
are considered amino acids for the purposes of the invention.
"Amino acid" also includes imino acid residues such as proline and
hydroxyproline. The side chains may be in either the (R) or the (S)
configuration. In the preferred embodiment, the amino acids are in
the (S) or L-configuration. If non-naturally occurring side chains
are used, non-amino acid substituents may be used, for example to
prevent or retard in vivo degradations.
[0024] In a preferred embodiment, the candidate bioactive agents
are naturally occuring proteins or fragments of naturally occuring
proteins. Thus, for example, cellular extracts containing proteins,
or random or directed digests of proteinaceous cellular extracts,
may be attached to beads as is more fully described below. In this
way libraries of procaryotic and eucaryotic proteins may be made
for screening against any number of targets. Particularly preferred
in this embodiment are libraries of bacterial, fungal, viral, and
mammalian proteins, with the latter being preferred, and human
proteins being especially preferred.
[0025] In a preferred embodiment, the candidate bioactive agents
are peptides of from about 2 to about 50 amino acids, with from
about 5 to about 30 amino acids being preferred, and from about 8
to about 20 being particularly preferred. The peptides may be
digests of naturally occuring proteins as is outlined above, random
peptides, or "biased" random peptides. By "randomized" or
grammatical equivalents herein is meant that each nucleic acid and
peptide consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic
acids, discussed below) are chemically synthesized, they may
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized candidate bioactive proteinaceous
agents.
[0026] The library should provide a sufficiently structurally
diverse population of randomized agents to effect a
probabilistically sufficient range of diversity to allow binding to
a particular target. Accordingly, an interaction library must be
large enough so that at least one of its members will have a
structure that gives it affinity for the target. Although it is
difficult to gauge the required absolute size of an interaction
library, nature provides a hint with the immune response: a
diversity of 10.sup.7-10.sup.8 different antibodies provides at
least one combination with sufficient affinity to interact with
most potential antigens faced by an organism. Published in vitro
selection techniques have also shown that a library size of
10.sup.7 to 10.sup.8 is sufficient to find structures with affinity
for the target. A library of all combinations of a peptide 7 to 20
amino acids in length, such as generally proposed herein, has the
potential to code for 20.sup.7 (10.sup.9) to 20.sup.20. Thus, with
libraries of 10.sup.7 to 10.sup.8 different molecules the present
methods allow a "working" subset of a theoretically complete
interaction library for 7 amino acids, and a subset of shapes for
the 20.sup.20 library. Thus, in a preferred embodiment, at least
10.sup.6, preferably at least 10.sup.7, more preferably at least
10.sup.8 and most preferably at least 10.sup.9 different sequences
are simultaneously analyzed in the subject methods. Preferred
methods maximize library size and diversity.
[0027] In one embodiment, the library is fully randomized, with no
sequence preferences or constants at any position. In a preferred
embodiment, the library is biased. That is, some positions within
the sequence are either held constant, or are selected from a
limited number of possibilities. For example, in a preferred
embodiment, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or
histidines for phosphorylation sites, etc., or to purines, etc.
[0028] In a preferred embodiment, the bias is towards peptides or
nucleic acids that interact with known classes of molecules. For
example, when the candidate bioactive agent is a peptide, it is
known that much of intracellular signaling is carried out via short
regions of polypeptides interacting with other polypeptides through
small peptide domains. For instance, a short region from the HIV-1
envelope cytoplasmic domain has been previously shown to block the
action of cellular calmodulin. Regions of the Fas cytoplasmic
domain, which shows homology to the mastoparan toxin from Wasps,
can be limited to a short peptide region with death-inducing
apoptotic or G protein inducing functions. Magainin, a natural
peptide derived from Xenopus, can have potent anti-tumour and
anti-microbial activity. Short peptide fragments of a protein
kinase C isozyme (.beta.PKC), have been shown to block nuclear
translocation of .beta.PKC in Xenopus oocytes following
stimulation. And, short SH-3 target peptides have been used as
psuedosubstrates for specific binding to SH-3 proteins. This is of
course a short list of available peptides with biological activity,
as the literature is dense in this area. Thus, there is much
precedent for the potential of small peptides to have activity on
intracellular signaling cascades. In addition, agonists and
antagonists of any number of molecules may be used as the basis of
biased randomization of candidate bioactive agents as well.
[0029] Thus, a number of molecules or protein domains are suitable
as starting points for the generation of biased randomized
candidate bioactive agents. A large number of small molecule
domains are known, that confer a common function, structure or
affinity. In addition, as is appreciated in the art, areas of weak
amino acid homology may have strong structural homology. A number
of these molecules, domains, and/or corresponding consensus
sequences, are known, including, but are not limited to, SH-2
domains, SH-3 domains, Pleckstrin, death domains, protease
cleavage/recognition sites, enzyme inhibitors, enzyme substrates,
Traf, etc. Similarly, there are a number of known nucleic acid
binding proteins containing domains suitable for use in the
invention. For example, leucine zipper consensus sequences are
known.
[0030] In a preferred embodiment, the candidate bioactive agents
are nucleic acids. By "nucleic acid" or "oligonucleotide" or
grammatical equivalents herein means at least two nucleotides
covalently linked together. A nucleic acid of the present invention
will generally contain phosphodiester bonds, although in some
cases, as outlined below, nucleic acid analogs are included that
may have alternate backbones, comprising, for example,
phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and
references therein; Letsinger, J. Org. Chem. 35:3800 (1970);
Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al.,
Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805
(1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and
Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate
(Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No.
5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc.
111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press), and peptide nucleic acid backbones and linkages
(see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem.
Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);
Carlsson et al., Nature 380:207 (1996), all of which are
incorporated by reference). Other analog nucleic acids include
those with positive backbones (Denpcy et al., Proc. Natl. Acad.
Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos.
5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991);
Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et
al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3,
ASC Symposium Series 580, "Carbohydrate Modifications in Antisense
Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,
Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al.,
J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996))
and non-ribose backbones, including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, "Carbohydrate Modifications in Antisense Research", Ed.
Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995)
pp169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of additional moieties such as labels, or to increase the
stability and half-life of such molecules in physiological
environments.
[0031] As will be appreciated by those in the art, all of these
nucleic acid analogs may find use in the present invention. In
addition, mixtures of naturally occurring nucleic acids and analogs
can be made. Alternatively, mixtures of different nucleic acid
analogs, and mixtures of naturally occuring nucleic acids and
analogs may be made.
[0032] Particularly preferred are peptide nucleic acids (PNA) which
includes peptide nucleic acid analogs. These backbones are
substantially non-ionic under neutral conditions, in contrast to
the highly charged phosphodiester backbone of naturally occurring
nucleic acids.
[0033] The nucleic acids may be single stranded or double stranded,
as specified, or contain portions of both double stranded or single
stranded sequence. The nucleic acid may be DNA, both genomic and
cDNA, RNA or a hybrid, where the nucleic acid contains any
combination of deoxyribo- and ribo-nucleotides, and any combination
of bases, including uracil, adenine, thymine, cytosine, guanine,
inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc. As
used herein, the term "nucleoside" includes nucleotides and
nucleoside and nucleotide analogs, and modified nucleosides such as
amino modified nucleosides. In addition, "nucleoside" includes
non-naturally occuring analog structures. Thus for example the
individual units of a peptide nucleic acid, each containing a base,
are referred to herein as a nucleoside.
[0034] As described above generally for proteins, nucleic acid
candidate bioactive agents may be naturally occuring nucleic acids,
random nucleic acids, or "biased" random nucleic acids. For
example, digests of procaryotic or eucaryotic genomes may be used
as is outlined above for proteins. Where the ultimate expression
product is a nucleic acid, at least 10, preferably at least 12,
more preferably at least 15, most preferably at least 21 nucleotide
positions need to be randomized, with more preferable if the
randomization is less than perfect. Similarly, at least 5,
preferably at least 6, more preferably at least 7 amino acid
positions need to be randomized; again, more are preferable if the
randomization is less than perfect.
[0035] In a preferred embodiment, the candidate bioactive agents
are organic moieties. In this embodiment, as is generlly described
in WO 94/24314, candidate agents are synthesized from a series of
substrates that can be chemically modified. "Chemically modified"
herein includes traditional chemical reactions as well as enzymatic
reactions. These substrates generally include, but are not limited
to, alkyl groups (including alkanes, alkenes, alkynes and
heteroalkyl), aryl groups (including arenes and heteroaryl),
alcohols, ethers, amines, aldehydes, ketones, acids, esters,
amides, cyclic compounds, heterocyclic compounds (including
purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines,
cephalosporins, and carbohydrates), steroids (including estrogens,
androgens, cortisone, ecodysone, etc.), alkaloids (including
ergots, vinca, curare, pyrollizdine, and mitomycines),
organometallic compounds, hetero-atom bearing compounds, amino
acids, and nucleosides. Chemical (including enzymatic) reactions
may be done on the moieties to form new substrates or candidate
agents which can then be tested using the present invention.
[0036] As will be appreciated by those in the art, it is possible
to screen more than one type of candidate agent at a time. Thus,
the library of candidate agents used in any particular assay may
include only one type of agent (i.e. peptides), or multiple types
(peptides and organic agents).
[0037] The candidate bioactive agents are covalently attached to
beads. By "beads" or "particles" or "solid supports" or
"microspheres" or grammatical equivalents herein is meant small
discrete particles, preferably but not required to be roughly
spherical, generally of about 3 to about 200 .mu.m in diameter. The
composition of the beads will vary, depending on the class of
candidate bioactive agent and the method of synthesis. Suitable
bead compositions include those used in peptide, nucleic acid and
organic moiety synthesis, including, but not limited to, glass,
polymers such as polystyrene, latex or cross-linked dextrans such
as Sepharose, cellulose, nylon, teflon, TentaGel, etc.
[0038] In a preferred embodiment, the beads may be labeled, either
with a fluorescent label, or preferably another type of label, for
example, binary tags that allow the subsequent identification of
the agent on the bead; see Maclean et al., PNAS USA 94:2805 (1997);
Ni et al., J. Med. Chem. 39:1601 (1996); Ni et al., Methods in
Enzymol. 267:261 (1996); and Gallop, Chemtracts-Organic Chemistry
7:172 (1994), all of which are incorporated by reference.
[0039] As is generally described herein, the candidate bioactive
agents may either be synthesized directly on the beads, or they may
be made and then attached after synthesis.
[0040] In a preferred embodiment, the candidate agents are
synthesized directly on the beads. As is known in the art, many
classes of chemical compounds are currently synthesized on solid
supports, such as peptides, organic moieties, and nucleic acids. It
is a relatively trivial matter to adjust the current synthetic
techniques to use beads. As will be appreciated by those in the
art, the literature contains numerous examples of the synthesis of
candidate agents (particularly libraries of candidate agents on
solid-phase supports; see for example Pavia et al. Bioorganic &
Medicinal Chemistry 1996 4(5):659-666; Liskamp et al., Bioorganic
& Medicinal Chemistry 1996 4(5):667-672; Tong et al.,
Bioorganic & Medicinal Chemistry 1996 4(5):693-698; Houghten et
al., Bioorganic & Medicinal Chemistry 1996 4(5):709-715, Freier
et al., Bioorganic & Medicinal Chemistry 1996 4(5):717-725;
Bolton et al., Tetrahedron Letters 1996 37(20) 3433-3436, all of
which are hereby expressly incorporated by reference).
[0041] In a preferred embodiment, it may be desirable to use
linkers to attach the candidate agents to the beads, to allow both
good attachment, sufficient flexibility to allow good interaction
with the target molecule, and to avoid undesirable binding
reactions. See Yu et al., Bioorganic & Medicinal Chemistry
Letters 1997 7(1) 95-98; Wennemers et al., Tett. Lett. p6413
(1994), both of which are incorporated by reference.
[0042] One advantage of the present invention is that it allows the
screening of intermediates in a synthetic pathway. That is, when
the candidate agents are synthesized on the beads, it is possible
to screen the beads at any point during the synthesis. Thus for
example, the initial chemical unit or substrate is attached to the
beads, and a first chemical modification reaction is done, to form
a set of derivatives different from the starting set. Chemical
modification in this context includes both chemical or enzymatic
reactions. These reactions can generally be categorized into
classes by the number of substrate and products. Thus, a first
class transforms a single substrate into a single product; for
example, an isomerization reaction. A second class joins two or
more substrates to form one product; a dehydration reaction joining
two nucleotides by an ester bond is an example. A third class
cleaves one substrate into two or more products; for example, the
cleavage of a protein by a protease. A fourth class transforms two
substrates into two products, for example by the transfer of a
reactive group from one substrate to another. All of these chemical
modification reactions can be the result of chemical reactions or
enzymatic ones, with "enzymatic" in this context including the use
of naturally or non-naturally occuring enzymes, as well as other
catalysts (such as catalytic surfaces or metal ligands).
[0043] The new moieties (i.e. starting materials after a chemical
modification) are then tested as candidate bioactive agents using
the methods of the present invention. Additional reactions can then
be done, and the beads retested. As will be appreciated by those in
the art, in some embodiments it may be useful to remove any beads
that contain bioactive agents that bind the target molecules, and
only do additional synthetic reaction steps on candidate agents
that do not bind the target. Alternatively, it may be desirable to
modify bioactive agents that do bind the target molecule in order
to generate bioactive agents that bind to the target with a higher
affinity, or to a related target, etc. In this embodiment, any
bound target molecules must be removed prior to further chemical
modification. Thus, the present invention provides a rapid and easy
way of screening intermediates in a given synthetic pathway.
[0044] In a preferred embodiment, the candidate agents are
synthesized first, and then covalently attached to the beads. As
will be appreciated by those in the art, this will be done
depending on the composition of the candidate agents and the beads.
The functionalization of solid support surfaces such as certain
polymers with chemically reactive groups such as thiols, amines,
carboxyls, etc. is generally known in the art. Generally, the
candidate agents are attached using functional groups on the
candidate agent. For example, candidate agents containing
carbohydrates may be attached to an amino-functionalized support;
the aldehyde of the carbohydrate is made using standard techniques,
and then the aldehyde is reacted with an amino group on the
surface. In an alternative embodiment, a sulfhydryl linker may be
used. There are a number of sulfhydryl reactive linkers known in
the art such as SPDP, maleimides, .alpha.-haloacetyls, and pyridyl
disulfides (see for example the 1994 Pierce Chemical Company
catalog, technical section on cross-linkers, pages 155-200,
incorporated herein by reference) which can be used to attach
cysteine containing proteinaceous agents to the support.
Alternatively, an amino group on the candidate agent may be used
for attachment to an amino group on the surface. For example, a
large number of stable bifunctional groups are well known in the
art, including homobifunctional and heterobifunctional linkers (see
Pierce Catalog and Handbook, pages 155-200). In an additional
embodiment, carboxy groups (either from the surface or from the
candidate agent) may be derivatized using well known linkers (see
the Pierce catalog). For example, carbodiimides activate carboxy
groups for attack by good nucleophiles such as amines (see
Torchilin et al., Critical Rev. Therapeutic Drug Carrier Systems,
7(4):275-308 (1991), expressly incorporated herein). Similarly, a
number of homo- and heterobifunctional agents are known for
amine-amine crosslinking, thiol-thiol crosslinking, amine-thiol
crosslinking, amine-carboxylic acid crosslinking, and carbohydrate
crosslinking to amines and thiols; see Molecular Probes Catalog,
1996, Sixth Edition, chapter 5, hereby incorporated by reference.
In addition, proteinaceous candidate agents may also be attached
using other techniques known in the art, for example for the
attachment of antibodies to polymers; see Slinkin et al., Bioconj.
Chem. 2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al.,
Bioconj. Chem. 3:323-327 (1992); King et al., Cancer Res.
54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem.
5:220-235 (1994), all of which are hereby expressly incorporated by
reference). It should be understood that the candidate agents may
be attached in a variety of ways, including those listed above.
What is important is that manner of attachment does not
significantly alter the functionality of the candidate agent; that
is, the candidate agent should be attached in such a flexible
manner as to allow its interaction with a target.
[0045] In general, it is desirable to have a library of candidate
agents attached to beads. By "library of candidate agents" herein
is meant generally at least about 10.sup.2 different compounds,
with at least about 10.sup.3 different compounds being preferred,
and at least about 10.sup.4, 10.sup.5 or 10.sup.6 different
compounds being particularly preferred.
[0046] In general, it is preferred that each bead contain a
multiplicity of candidate agents. That is, each bead will contain
at least about 10 candidate agents, with at least about 100 being
preferred, and at least about 1000 being especially preferred.
[0047] As will be appreciated by those in the art, each bead may
contain one type of candidate agent, or more than one. That is, in
a preferred embodiment, any single bead contains a single type of
candidate bioactive agent. This may be preferred for a variety of
reasons, including synthetic considerations, ease of
characterization of downstream "hits", and fluorescent detection
limits (that is, the number of fluorescent targets that should be
bound to a bead to allow FACS sorting).
[0048] Alternatively, (for example when libraries of naturally
occuring compounds are attached to beads), each bead may contain
more than one type of candidate agent. In this embodiment, as is
more fully outlined herein, it will generally be desirable to
"amplify" the fluorescent signal (i.e. have more than one
fluorescent label per target) to facilitate detection.
[0049] In a preferred embodiment, there are a number of beads that
each contain a single candidate agent. That is, there are a number
of beads each containing a particular candidate agent. Thus, at
least about 100 beads per candidate agent are used, with at least
about 1000 being preferred and at least about 10,000 to 100,000
being especially preferred.
[0050] Thus, the library of candidate bioactive agents are
contained upon a plurality of beads.
[0051] Once generated, the library of beads containing a library of
covalently attached candidate agents is added to at least a first
population of a first target molecule. By "target molecule" herein
is meant a molecule for which an interaction is sought; this term
will be generally understood by those in the art. Suitable target
molecules include, but are not limited to, proteins such as
receptors, enzymes, cell-surface receptors, G-protein coupled
receptors, ion channels, transport proteins, transcription factors,
vesicle proteins, adhesion proteins, etc.
[0052] The target molecule comprises a first labeling moiety.
Either the first labeling moiety comprises a fluorescent label, or
a second labeling moiety is used, that is fluorescent and will bind
to the first labeling moiety.
[0053] In a preferred embodiment, the first labeling moiety
comprises at least a first fluorescent label. Suitable fluorescent
labels include, but are not limited to, fluorescein, rhodamine,
tetramethylrhodamine, eosin, erythrosin, coumarin,
methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,
Cascade Blue.TM., and Texas Red. Suitable optical dyes are
described in the 1996 Molecular Probes Handbook by Richard P.
Haugland, hereby expressly incorporated by reference.
[0054] In a preferred embodiment, the fluorescent label is
functionalized to facilitate covalent attachment, as is generally
outlined above for the attachment of candidate agents to surfaces.
Thus, a wide variety of fluorescent labels are commercially
available which contain functional groups, including, but not
limited to, isothiocyanate groups, amino groups, haloacetyl groups,
maleimides, succinimidyl esters, and sulfonyl halides, all of which
may be used to covalently attach the fluorescent label to a second
molecule, as is described herein. The choice of the functional
group of the fluorescent label will depend on the site of
attachment to either a linker, as outlined below, the first
labeling moiety, the target molecule, or the second labeling
moiety.
[0055] The covalent attachment of the fluorescent label may be
either direct or via a linker. In one embodiment, the linker is a
relatively short coupling moiety, that is used to attach the
molecules. A coupling moiety may be synthesized directly onto a
candidate agent for example, and contains at least one functional
group to facilitate attachment of the fluorescent label.
Alternatively, the coupling moiety may have at least two functional
groups, which are used to attach a functionalized candidate agent
to a functionalized fluorescent label, for example. In an
additional embodiment, the linker is a polymer. In this embodiment,
covalent attachment is accomplished either directly, or through the
use of coupling moieties from the agent or label to the polymer. In
a preferred embodiment, the covalent attachment is direct, that is,
no linker is used. In this embodiment, the candidate agent
preferably contains a functional group such as a carboxylic acid
which is used for direct attachment to the functionalized
fluorescent label. Thus, for example, for direct linkage to a
carboxylic acid group of a candidate agent, amino modified or
hydrazine modified fluorescent labels will be used for coupling via
carbodiimide chemistry, for example using
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as is known in
the art (see Set 9 and Set 11 of the Molecular Probes Catalog,
supra; see also the Pierce 1994 Catalog and Handbook, pages T-155
to T-200, both of which are hereby incorporated by reference). In
one embodiment, the carbodiimide is first attached to the
fluorescent label, such as is commercially available.
[0056] Thus, in a preferred embodiment, a fluorescent label is
attached, either directly or via a linker, to the candidate agents
and thus serves as a first labeling moiety. Alternatively, in a
preferred embodiment, the first labeling moiety comprises a first
partner of a binding pair, which may or may not be fluorescent, and
a second labeling moiety, comprising the second partner of a
binding pair, and at least one fluorescent label, as defined
above.
[0057] Suitable binding pairs include, but are not limited to,
antigens/antibodies, including digoxigenin/antibody, dinitrophenyl
(DNP)/anti-DNP, dansyl-X/anti-dansyl, fluorescein/anti-fluorescein,
lucifer yellow/anti-lucifer yellow, rhodamine/anti-rhodamine; and
biotin/avidin (or biotin/strepavidin). Preferred binding pairs
(i.e. first and second labeling moieties) generally have high
affinities for each other, sufficient to withstand the shear forces
during FACS sorting.
[0058] Generally, in a preferred embodiment, the smaller of the
binding partners serves as the first labeling moiety, as steric
considerations in agent:target binding may be important. Thus,
preferred first labeling moieties (when second labeling moieties
are used), include, but are not limited to, haptens such as biotin,
etc. Biotinylation of target molecules is well known, for example,
a large number of biotinylation agents are known, including
amine-reactive and thiol-reactive agents, for the biotinylation of
proteins, nucleic acids, carbohydrates, carboxylic acids; see
chapter 4, Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby
incorporated by reference. Similarly, a large number of
haptenylation reagents are also known; supra.
[0059] In this embodiment, attachment of the first labeling moiety
to the candidate agents will be done as is generally appreciated by
those in the art, and may include techniques outlined above for the
incorporation of fluorescent labels.
[0060] In this embodiment, a second labeling moiety that comprises
at least a first fluorescent label is used. The fluorescent labels
are generally attached as outlined above. The second labeling
moiety, i.e. the other partner of the binding pair, has at least
one fluorescent label, with at least 5 being preferred and at least
10 being particularly preferred. This is determined on the basis of
the sensitivity of the FACS sorting. In general, 10 to 100 fluores
per sorting event are needed; i.e. per bead, with from about 20 to
100 being preferred, and from 30 to 90 being especially preferred.
This can be accomplished by amplifying the signal per target, i.e.
have each second label comprise multiple fluores, or by having a
high density of target binding per bead; or a combination of both.
Thus, in some situations, binding of ten targets to a single bead,
each containing at least one to 10 fluores may be done.
[0061] In a preferred embodiment, all the target molecules contain
the same fluorescent label (that is, all of either the first or the
second labeling moieties comprise a single type of fluorescent
label). In an alternative embodiment, the target molecule
population is divided into at least two subpopulations, each
comprising a different fluorescent label. This may be particularly
preferred to reduce false positives; that is, only beads comprising
both labels (i.e. beads with a single candidate agent type that
bind targets with both labels) will constitute "real" interactions.
In a preferred embodiment, up to four different labels may be used
in the current FACS systems, with eight being possible soon.
[0062] In one embodiment, the target molecules are also bound to
beads. In a preferred embodiment, the target molecules are attached
to the beads using preferably flexible linkers, to allow for
interaction with bead-bound agents. In this embodiment, a preferred
system utilizes fluorescent beads; that is, the bead to which the
target molecules is attached can be fluorescent, thus serving as
the first or second labeling moiety. See for example the Molecular
Probes catalog, supra, chapter 6, hereby incorporated by
reference.
[0063] The beads containing the candidate agents are added to the
target molecules under reaction conditions that favor agent-target
interactions. Generally, this will be physiological conditions.
Incubations may be performed at any temperature which facilitates
optimal activity, typically between 4 and 40.degree. C. Incubation
periods are selected for optimum activity, but may also be
optimized to facilitate rapid high through put screening. Typically
between 0.1 and 1 hour will be sufficient. Excess reagent is
generally removed or washed away.
[0064] A variety of other reagents may be included in the assays.
These include reagents like salts, neutral proteins, e.g. albumin,
detergents, etc which may be used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., may be used. The mixture of components
may be added in any order that provides for the requisite
binding.
[0065] In a preferred embodiment, the beads containing the
candidate agents are added to a single target molecule population,
i.e. where the target molecules are all the same. In an additional
preferred embodiment, the beads can be added to more than one
target molecule population at a time, with each target molecule
preferably containing, although it is not required, a different
fluorescent label.
[0066] In a preferred embodiment, the target molecules comprising
the first labeling moieties are added first, and allowed to react
under favorable binding conditions for a period of time. The beads
are then removed from the reaction mixture, optionally but
preferably washed or rinsed one or more times to remove excess
reagents including target molecules, and either sorted, or the
second labeling moiety, if used, is added. Washing or rinsing the
beads will be done as will be appreciated by those in the art, and
may include the use of filtration, centrifugation, the application
of a magnetic field, electrostatic interactions for charged beads,
adhesion, etc. When second labeling moieties are used, they are
preferably added after excess non-bound target molecules are
removed, in order to reduce non-specific binding; however, under
some circumstances, all the components may be added
simultaneously.
[0067] The beads are then sorted using fluorescent-activated cell
sorting (FACS). In general, K.sub.Ds of .ltoreq.1 .mu.M are
preferred, to allow for retention of binding in the presence of the
shear forces present in FACS sorting. In a preferred embodiment,
the beads are sorted at very high speeds, for example greater than
about 5,000 sorting events per sec, with greater than about 10,000
sorting events per sec being preferred, and greater than about
25,000 sorting events per second being particularly preferred, with
speeds of greater than about 50,000 to 100,000 being especially
preferred.
[0068] The sorting results in a population of non-fluorescent beads
and at least one population of fluorescent beads, depending on how
many fluorescent labels were used. The presence of at least one
fluorescent bead is indicative that at least one candidate
bioactive agent is a bioactive agent that binds to the target
molecule.
[0069] Once a fluorescent bead, i.e. a bead containing at least one
bioactive agent that is bound to the target, is isolated, a number
of things may be done.
[0070] In a preferred embodiment, the chacterization of the
bioactive agent is done. This will proceed as will be appreciated
by those in the art, and generally includes an analysis of the
structure, identity, binding affinity and function of the
agent.
[0071] In one embodiment, the candidate agent is attached to the
bead in such a manner as to allow subsequent cleavage. Thus, in
this embodiment, the candidate agent may be cleaved off and
analyzed, for example via mass spectroscopy, to elucidate the
structure of the bioactive agent. Alternatively, binary tags such
as those outlined above may be used.
[0072] Generally, once identified, the bioactive agent is
resynthesized and combined with the target molecule to verify the
binding. In addition, once binding bioactive agents are found, they
may be tested in functional screens to determine their effect on
the target molecule. These functional screens will be done
depending on the target molecule, as will be appreciated by those
in the art.
[0073] In a preferred embodiment, either the non-reactive beads
(i.e. the non-fluorescent beads), or the fluorescent beads (to
which fluorescent target is bound), or both, can be subjected to
altered experimental conditions and resorted. This may be done, for
example, to quantify or alter the binding affinity of the bioactive
agent for the target. Thus, for example, changes in pH,
temperature, buffer or salt concentration, etc. In a preferred
embodiment, the pH is changed, generally by increasing or
decreasing the pH, usually by from about 0.5 to about 3 pH units.
Alternatively, the temperature is altered, with increases or
decreases of from about 5.degree. C. to about 30.degree. C. being
preferred. Similarly, the salt concentration may be modified, with
increases or decreases of from about 0.1 M to about 2 M being
preferred.
[0074] In a preferred embodiment, either the non-reactive beads
(i.e. the non-fluorescent beads), or the fluorescent beads (to
which fluorescent target is bound), or both, can be treated such
that further chemical modifications are done. As outlined herein,
the present invention is useful to test the products from each
chemical step in a synthetic reaction against one or more target
molecules, for example in combinatorial chemical library synthesis.
Thus, at any point in a synthetic scheme, beads containing the
products maybe tested using the present methods. This may require
the washing or buffer exchange of the beads as is outlined herein.
The beads are sorted, and either or both of the fluorescent and
non-fluorescent beads may be returned to a reaction vessel for
further chemical modifications, as outlined above.
[0075] In a preferred embodiment, the affinity and specificity of
the binding reaction may be tested using a known binding agent,
i.e. a competitor. Thus generally, the population of fluorescent
beads (i.e. beads to which a fluorescent target is bound), is
subjected to further steps. A binding molecule is added to the
fluorescent beads. By "binding molecule" or "competitor moiety" or
grammatical equivalents herein is meant a molecule known to bind to
the target molecule, including, but not limited to, antibodies,
peptides and nucleic acids.
[0076] In a preferred embodiment, the competitor is also labeled,
preferably with a different fluorescent label than the target. This
will allow the detection of binding agents that either bind to the
same binding site as the competitor (in which case only one label
will be detected), or that bind to a different binding site than
the competitor (in which case two labels should be detected).
[0077] In a preferred embodiment, either the non-reactive beads
(i.e. the non-fluorescent beads), or the fluorescent beads (to
which fluorescent target is bound), or both, can be added to a
second population of second target molecules. As is outlined
herein, this may be done either simultaneously with the first
target molecule population or sequentially. In a preferred
embodiment, particularly when two target molecules are analyzed at
the same time, the second target molecule utilizes a different
fluorescent label than the first target molecule, although this is
not required. Additionally embodiments utilize third, fourth, etc.
populations of target molecules.
[0078] As will be appreciated by those in the art, the ability to
reuse the candidate agent library, on the beads, is a significant
advantage. As will be appreciated by those in the art, when
sequential analysis is done, it is preferable to remove any bound
target molecules from the beads, for example by increasing the salt
concentration, pH or temperature, or by adding a competitor, to
remove any target molecules bound to the beads. All references
cited herein are incorporated by reference.
* * * * *