U.S. patent application number 10/992334 was filed with the patent office on 2005-05-05 for factorial chemical libraries.
This patent application is currently assigned to AFFYMETRIX, INC.. Invention is credited to Fodor, Stephen P.A., Stryer, Lubert.
Application Number | 20050095638 10/992334 |
Document ID | / |
Family ID | 23267961 |
Filed Date | 2005-05-05 |
United States Patent
Application |
20050095638 |
Kind Code |
A1 |
Fodor, Stephen P.A. ; et
al. |
May 5, 2005 |
Factorial chemical libraries
Abstract
A method and library for determining the sequence of monomers in
a polymer which is complementary to a receptor. The method provides
for formation of pooled (6) and separate (10, 12) products.
Separate products are subjected only to subsequent pooled coupling
steps. Each pooled product is subsequently divided for formation of
pooled and separate products. The resulting polymer library
includes groups of polymer products. A first group of products (42)
is used to identify the monomer at a first location in a polymer
that is complementary to a receptor. A second group of products
(44) is used to identify the monomer at a second location in a
polymer that is complementary to a receptor.
Inventors: |
Fodor, Stephen P.A.; (Palo
Alto, CA) ; Stryer, Lubert; (Stanford, CA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE, N.W.
WASHINGTON
DC
20004
US
|
Assignee: |
AFFYMETRIX, INC.
|
Family ID: |
23267961 |
Appl. No.: |
10/992334 |
Filed: |
November 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10992334 |
Nov 19, 2004 |
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08325457 |
Oct 28, 1994 |
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6864048 |
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08325457 |
Oct 28, 1994 |
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PCT/US93/04145 |
Apr 28, 1993 |
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Current U.S.
Class: |
435/6.12 ;
435/6.1; 435/7.1; 436/518 |
Current CPC
Class: |
C40B 40/00 20130101;
C12N 15/1034 20130101; C07K 1/047 20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 436/518 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/543 |
Claims
What is claimed is:
1. A polymer library screening kit comprising families of polymers
X.sub.3-X.sub.2pX.sub.1p, X.sub.3p-X.sub.2-X.sub.1p and
X.sub.3p-X.sub.2p-X.sub.1 wherein: X.sub.3p-X.sub.2p-X.sub.1
comprises a collection of at least first and second polymer
mixtures, said first polymer mixture having a first monomer in a
first position of polymer molecules therein, and different monomers
in second and third positions of said polymer molecules therein,
and wherein said second polymer mixture has a second monomer in
said first position of polymer molecules therein, and different
monomers in second and third positions of said polymer molecules
therein; X.sub.3p-X.sub.2-X.sub.1p comprises a collection of at
least third and fourth polymer mixtures, said third polymer mixture
having a third monomer in said second position and said fourth
polymer mixture having a fourth monomer in said second position,
each of said third and fourth polymer mixtures having different
monomers in said first and third positions; and
X.sub.3-X.sub.2p-X.sub.1p comprises a collection of at least fifth
and sixth polymer mixtures, said fifth polymer mixture having a
fifth monomer in said third position and said sixth polymer mixture
having a sixth monomer in said third position, each of said fifth
and sixth polymer mixtures having different monomers in said first
and second positions, wherein said first, third, and fourth
monomers are the same or different and said second, fourth, and
fifth monomers are the same or different.
2. The polymer library as recited in claim 1 wherein each of said
polymer mixtures are selected from the group consisting of mixtures
of peptides and mixtures of oligonucleotides.
3. The polymer library as recited in claim 1 wherein said polymers
comprise at least four monomers.
4. The polymer library as recited in claim 1 further comprising
labelled receptor molecules, and means for identifying mixtures of
said polymers to which said receptor molecules are bound.
5. The polymer library as recited in claim 9 wherein said receptor
molecules are labelled with a fluorescein label.
6. The polymer library as recited in claim 1 wherein said polymers
are coupled to a solid substrate.
7. A method of identifying first and second monomers in a polymer
that is complementary to a receptor comprising the steps of:
coupling first and second monomers in a first basis set to
individual substrates and mixing substrates to form first pooled
products; coupling said first and second monomers from said first
basis set to individual substrates, and not mixing said substrates
to form at least first and second separate products; separately
coupling first and second monomers from a second basis set to
substrates from said first pooled products and not mixing said
substrates to form at least third and fourth separate products;
coupling said first and second monomers from said second basis set
to individual substrates from said first separate products and
mixing said substrates to form second pooled products; coupling
said first and second monomers from said second basis set to
individual substrates from said second separate products to form
third pooled products; and exposing a receptor to said third and
fourth separate products to identify a second monomer in a polymer
which is complementary to a receptor, and exposing said second and
third pooled products to said receptor to identify a first monomer
in a polymer which is complementary to said receptor.
8. The method as recited in claim 7 wherein said step of exposing
to a receptor is preceded by the step of performing additional
steps of coupling and mixing to said second pooled products and
said third pooled products.
9. The method as recited in claim 7 further comprising the step of
mixing a portion of said third and fourth separate products to form
fourth pooled products.
10. The method as recited in claim 9 further comprising the step of
separately coupling monomers from a third basis set to said fourth
pooled products.
11. The method as recited in claim 7 wherein said monomers are
amino acids.
12. The method as recited in claim 7 wherein said monomers are
nucleotides.
13. The method as recited in claim 7 wherein said steps are
repeated to screen polymers having at least three monomers
therein.
14. The method as recited in claim 7 wherein at least one of said
first and second monomers cannot be determined unambiguously,
further comprising the steps of: synthesizing an array of polymers,
said potential complementary polymers using a light-directed
synthesis technique; and detecting binding of said receptor to said
potential complementary polymers.
15. A library of polymers to be used for identification of a
receptor complementary to at least one of said polymers comprising:
a first set of polymers, said first set of polymers having a first
monomer in a first position, and a plurality of different monomers
at a second position; and a second set of polymers, isolated from
said first set of polymers, said second set of polymers having a
second monomer in said second position, and a plurality of
different monomers in said first position.
16. A library of polymers as recited in claim 15 further
comprising: a third set of polymers, isolated from said first and
second sets of polymers, said third set of polymers having a third
monomer in said first position and a plurality of different
monomers at said second position.
17. A library of polymers as recited in claim 16 further
comprising: a fourth set of polymers, isolated from said first,
second, and third sets of polymers, said fourth set of polymers
having a fourth monomer in said second position and a plurality of
different monomers in said first position.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to the field of polymer
screening. More specifically, in one embodiment the invention
provides an improved polymer library and method of using the
library to identify a polymer sequence that is complementary to a
receptor.
[0002] Many assays are available for measuring the binding affinity
of receptors and ligands, but the information which can be gained
from such experiments is often limited by the number and type of
ligands which are available. Small peptides are an exemplary system
for exploring the relationship between structure and function in
biology. When the twenty naturally occurring amino acids are
condensed into peptides they form a wide variety of
three-dimensional configurations, each resulting from a particular
amino acid sequence and solvent condition. The number of possible
pentapeptides of the 20 naturally occurring amino acids, for
example, is 20.sup.5 or 3.2 million different peptides. The
likelihood that molecules of this size might be useful in
receptor-binding studies is supported by epitope analysis studies
showing that some antibodies recognize sequences as short as a few
amino acids with high specificity.
[0003] Prior methods of preparing large numbers of different
oligomers have been painstakingly slow when used at a scale
sufficient to permit effective rational or random screening. For
example, the "Merrifield" method, described in Atherton et al.,
"Solid Phase Peptide Synthesis," IRL Press, (1989), incorporated
herein by reference for all purposes, has been used to synthesize
peptides on a solid support such as pins or rods. The peptides are
then screened to determine if they are complementary to a receptor.
Using the Merrifield method, it is not economically practical to
screen more than a few peptides in a day.
[0004] Similar problems are encountered in the screening of other
polymers having a diverse basis set of monomers. For example,
various methods of oligonucleotide synthesis such as the
phosphite-triester method and the phosphotrieseter method,
described in Gait, "Oligonucleotide Synthesis," IRL Press, (1990),
incorporated herein by reference for all purposes, have similar
limitations when it is desired to synthesize many diverse
oligonucleotides for screening.
[0005] To screen a larger number of polymer sequences, more
advanced techniques have been disclosed. For example, Pirrung et
al., WO 90/15070, incorporated herein by reference for all
purposes, describes a method of synthesizing a large number of
polymer sequences on a solid substrate using light directed
methods. Dower et al., U.S. application Ser. No. 07/762,522, also
incorporated by reference herein for all purposes, describes a
method of synthesizing a library of polymers and a method of use
thereof. The polymers are synthesized on beads, for example. A
first monomer is attached to a pool of beads. Thereafter, the pool
of beads is divided, and a second monomer is attached. The process
is repeated until a desired, diverse set of polymers is
synthesized.
[0006] Other methods of synthesizing and screening polymers have
also been proposed. For example, Houghten et al., "Generation and
Use of Synthetic Peptide Combinatorial Libraries for Basic Research
and Drug Discovery," Nature (1991) 354:84-86, discuss a method of
generating peptide libraries that are used for screening the
peptides for biological activity (see also, Houghton et al., "The
Use of Synthetic Peptide Combinatorial Libraries for the
Identification of Bioactive Peptides," Peptide Research (1992)
5:351-358). Houghten synthesized a peptide combinatorial library
(SPCL) composed of some 34.times.10.sup.6 hexapeptides and screened
it to identify antigenic determinants that are recognized by a
monoclonal antibody. Furka et al., "General Method for Rapid
Synthesis of Multicomponent Peptide Mixtures," Int. J. Peptide
Protein Res. (1991) 37:487-493, discusses a method of synthesizing
multicomponent peptide mixtures. Furka proposed pooling as a
general method for the rapid synthesis of milticomponent peptide
mixtures and illustrated its application by synthesizing a mixture
of 27 tetrapeptides and 180 pentapeptides. Lam et al., "A new type
of synthetic peptide library for identifying ligand-binding
activity," Nature (1991) 354:82-84 used pooling to generate a
pentapeptide bead library that was screened for binding to a
monoclonal antibody. Blake et al. "Evaluation of Peptide Libraries:
An Interative Strategy To Analyze the Reactivity of Peptide
Mixtures With Antibodies," Bioconjugate Chem. (1992) 3:510-513
discusses, the screening of presumed mixtures of 50,625
tetrapeptides and 16,777,216 hexpeptides to select epitopes
recognized by specific antibodies.
[0007] Lam's synthetic peptide library consists of a large number
of beads, each bead containing peptide molecules of one kind. Beads
that bind a target (e.g., an antibody or strepavidin) are rendered
colored or fluorescent. Lam reports that several million beads
distributed in 10-15 petri dishes can be screened with a low-power
dissecting microscope in an afternoon. Positive beads are washed
with 8M guanidine hydrochloride to remove the target protein and
then sequenced. The 100-200 .mu.m diameter beads contain 50-200
pmol of peptide, putatively well above their 5 pmol sensitivity
limit. Three pentapeptide beads were sequenced daily. The essence
of Lam's method is that the identity of positive beads is
established by direct sequencing.
[0008] Houghten et al. use a different approach to identify peptide
sequences that are recognized by an antibody. Using the
nomenclature described herein, Houghten et al. screened an
X.sub.6X.sub.5X.sub.4pX.sub- .3pX.sub.2pX.sub.1p library and found
that the mixture DVX.sub.4pX.sub.3pX.sub.2pX.sub.1p had greatest
potency in their inhibition assay. Houghten then synthesized a
DVX.sub.4X.sub.3pX.sub.2pX.- sub.1p library and identified the most
potent amino acid in the third position. After three more
iterations, they found that DVPDYA binds to the antibody with a
K.sub.d of 30 nM. The essence of Houghten's method is recursive
retrosynthesis, in which the number of pooled positions decreases
by one each iteration.
[0009] Blake et al. used a "bogus coin strategy" to guide them to a
preferred amino acid sequence. In this strategy a basis set of
monomers (15 amino acids) is first divided into three groups. Blake
et al. chose A, L, V, F, Y (subgroup .alpha.), G, S, P, D, E
(subgroup .beta.), and K, R, H, N, Q (subgroup .gamma.). By
adjusting the "weighting of the subgroups at each position in the
polymer sequence, and then testing the activity of the weighted
polymer against an unweighted polymer, one subgroup was selected
for each monomer position in the sequence. In an experiment
conducted by Blake et al., a complete collection of tetramers
X.sub.1PX.sub.2PX.sub.3PX.sub.4P was reduced to
.alpha..sub.1.alpha..sub.- 2 .gamma..sub.3.alpha..sub.4 by four
inhibition experiments. Then the subgroups .alpha. and .gamma. were
each further subdivided into three groups of amino acid which were
used to synthesize four more collections of weighted polymers.
Inhibition studies with each of these collections suggested an
epitope (F or Y).sub.1 (A or L).sub.2 (K or R).sub.3 (F or
Y).sub.4. One more iteration gave the desired epitope FLRF.
[0010] While meeting with some success, prior methods have also met
with certain limitations. For example, it is sometimes desirable to
avoid the use of the equipment necessary to conduct light directed
techniques. Also, some prior methods have not produced the desired
amount of diversity as efficiently as would be desired.
[0011] From the above, it is seen that an improved method and
apparatus for synthesizing a diverse collection of chemical
sequences is desired.
SUMMARY OF THE INVENTION
[0012] An improved polymer library and method of screening diverse
polymers is disclosed. The system produces libraries of polymers in
an efficient manner, and utilizes the libraries for identification
of the monomer sequence of polymers which exhibit significant
binding to a ligand.
[0013] According to one aspect of the invention, a library of
polymers is formed using "pooled" and "unpooled" (or "separate")
coupling steps. In the pooled steps, each of the monomers from a
basis set of monomers is coupled to the terminus of a growing chain
of monomers on a plurality of previously mixed solid substrates.
The mixed substrates are divided for coupling of each individual
monomer in a basis set. In separate steps, the substrates are not
intermixed from a previous coupling step, and each of the monomers
in a basis set is separately coupled to the terminus of a growing
chain of monomers on a plurality of the unmixed substrates.
[0014] According to one preferred aspect of the invention, pooled
steps and unpooled steps are ordered such that the identification
of a monomer sequence which binds to a receptor can be readily
identified from the library. For example, according to one
preferred embodiment of the invention, several groups of products
are derived from the synthesis steps. Each group is used to
identify the monomer at a specific position in the polymer
chain.
[0015] According to most preferred aspects of the invention, the
library is constructed using an ordered series of coupling steps in
which products resulting from a separate step are, thereafter, only
subjected to pooled coupling steps. Products resulting from a
pooled coupling step which have not been previously subjected to an
unpooled step are always divided for pooled and unpooled coupling.
This ordered series of steps results in a relatively small number
of coupling steps, but still allows for identification of the
monomer sequence of a polymer which is complementary to a receptor
of interest. For example, a first group of products is used to
identify the monomer at a first location in a polymer that is
complementary to a receptor. A second group of products is used to
identify the monomer at a second location in a polymer that is
complementary to a receptor.
[0016] Accordingly, in one embodiment of the invention provides a
polymer library screening kit. The kit includes families of
polymers X.sub.3-X.sub.2p-X.sub.1pP, X.sub.3p-X.sub.2-X.sub.1p, and
X.sub.3p-X.sub.2p-X.sub.1 wherein X.sub.3p-X.sub.2p-X.sub.1
comprises a collection of at least first and second polymer
mixtures, the first polymer mixture having a first monomer in a
first position of polymer molecules therein, and different monomers
in second and third positions of the polymer molecules therein, and
wherein the second polymer mixture has a second monomer in the
first position of polymer molecules therein, and different monomers
in second and third positions of the polymer molecules therein;
X.sub.3p-X.sub.2-X.sub.1p comprises a collection of at least third
and fourth polymer mixtures, the third polymer mixture having a
third monomer in the second position and the fourth polymer mixture
having a fourth monomer in the second position, each of the third
and fourth polymer mixtures having different monomers in the first
and third positions; and X.sub.3-X.sub.2p-X.sub.1p comprises a
collection of at least fifth and sixth polymer mixtures, the fifth
polymer mixture having a fifth monomer in the third position and
the sixth polymer mixture having a sixth monomer in the third
position, each of the fifth and sixth polymer mixtures having
different monomers in the first and second positions, wherein the
first, third, and fourth monomers are the same or different and the
second, fourth, and fifth monomers are the same or different.
[0017] A method of identifying first and second monomers in a
polymer that is complementary to a receptor is also provided. The
method includes the steps of coupling first and second monomers in
a first basis set to individual substrates and mixing substrates to
form first pooled products; coupling the first and second monomers
from the first basis set to individual substrates, and not mixing
the substrates to form at least first and second separate products;
separately coupling first and second monomers from a second basis
set to substrates from the first pooled products and not mixing the
substrates to form at least third and fourth separate products, the
second basis set being the same or different than the first basis
set; coupling the first and second monomers from the second basis
set to individual substrates from the first separate products and
mixing the substrates to form second pooled products; coupling the
first and second monomers from the second basis set to individual
substrates from the second separate products to form third pooled
products; and exposing a receptor to the third and fourth separate
products to identify a second monomer in a polymer which is
complementary to a receptor, and exposing the second and third
pooled products to the receptor to identify a first monomer in a
polymer which is complementary to a receptor.
[0018] A polymer screening technique using factoring is also
disclosed.
[0019] A further understanding of the nature and advantages of the
inventions herein may be realized by reference to the remaining
portions of the specification and the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1a and 1b are schematic diagrams of specific
embodiments of the invention;
[0021] FIG. 2 illustrates a simple reaction graph;
[0022] FIG. 3 illustrates a reaction graph with pooled and separate
products;
[0023] FIG. 4 illustrates a simplified reaction graph;
[0024] FIGS. 5a, 5b, and 5c illustrate a family of pooled
syntheses;
[0025] FIG. 6 illustrates a reaction graph for forming the products
X.sub.3pX.sub.2X.sub.1p;
[0026] FIG. 7 illustrates a reaction graph for all 64
trinucleotides;
[0027] FIG. 8 illustrates the synthesis of AAT, TGC, TGT, GTA, GTG,
and CCG;
[0028] FIG. 9 provides an alternative representation of the
invention;
[0029] FIGS. 10a, 10b, and 10c illustrate a recursive
retrosynthesis embodiment of the invention;
[0030] FIGS. 11a, 11b, and 11c illustrate a combinatorial synthesis
chamber of the invention; and
[0031] FIGS. 12 illustrates a polymer library according to one
embodiment of the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Contents
[0032] I. Terminology
[0033] II. Overall Description
[0034] III. Polynomial Factoring Applied to Screening
[0035] IV. Conclusion
[0036] I. Terminology
[0037] Ligand: A ligand is a molecule that is recognized by a
particular receptor. Examples of ligands that can be investigated
by this invention include, but are not restricted to, agonists and
antagonists for cell membrane receptors, toxins and venoms, viral
epitopes, hormones (e.g., opiates, steroids, etc.), hormone
receptors, peptides, enzymes, enyme substrates, cofactors, drugs,
lectins, sugars, oligonucleotides, nucleic acids, oligosarcharides,
proteins, and monoclonal antibodies.
[0038] Monomer: A member of the set of small molecules which are or
can be joined together to form a polymer. The set of monomers
includes but is not restricted to, for example, the set of common
L-amino acids, the set of D-amino acids, the set of synthetic
and/or natural amino acids, the set of nucleotides and the set of
pentoses and hexoses, as well as subsets thereof. The particular
ordering of monomers within a polymer is referred to herein as the
"sequence" of the polymer. As used herein, monomers refers to any
member of a basis set for synthesis of a polymer. For example,
dimers of the 20 naturally occurring L-amino acids form a basis set
of 400 monomers for synthesis of polypeptides. Different basis sets
of monomers may be used at successive steps in the synthesis of a
polymer. Furthermore, each of the sets may include protected
members which are modified after synthesis. The invention is
described herein primarily with regard to the preparation of
molecules containing sequences of monomers such as amino acids, but
could readily be applied in the preparation of other polymers. Such
polymers include, for example, both linear and cyclic polymers of
nucleic acids, polysaccharides, phospholipids, and peptides having
either .alpha.-, .beta.-, or .omega.-amino acids, heteropolymers in
which a known drug is covalently bound to any of the above,
polynucleotides, polyurethanes, polyesters, polyciubonates,
polyureas, polyamides, polyethyleneimines, polyarylene sulfides,
polysiloxanes, polyimides, polyacetates, or other polymers which
will be apparent upon review of this disclosure. Such polymers are
"diverse" when polymers having different monomer sequences are
formed at different predefined regions of a substrate. Methods of
cyclization and polymer reversal of polymers which may be used in
conjunction with the present invention are disclosed in copending
application Ser. No. 796,727, filed Nov. 22, 1991 entitled "POLYMER
REVERSAL ON SOLID SURFACES," incorporated herein by reference for
all purposes. The "position" of a monomer in a polymer refers to
the distance, by number of monomers, from a terminus or other
reference location on a polymer.
[0039] Peptide: A polymer in which the monomers are alpha amino
acids and which are joined together through amide bonds,
alternatively referred to as a polypeptide. In the context of this
specification it should be appreciated that the amino acids may be
the L-optical isomer or the D-optical isomer. Peptides are often
two or more amino acid monomers long, and often more than 20 amino
acid monomers long. Standard abbreviations for amino acids are used
(e.g., P for proline). These abbreviations are included in Stryer,
Biochemistry, Third Ed., 1988, which is incorporated herein by
reference for all purposes.
[0040] Receptor: A molecule that has an affinity for a given
ligand. Receptors may be naturally-occurring or manmade molecules.
Also, they can be employed in their unaltered state or as
aggregates with other species. Receptors may be attached,
covalently or noncovalently, to a binding member, either directly
or via a specific binding substance. Examples of receptors which
can be employed by this invention include, but are not restricted
to, antibodies, cell membrane receptors, monoclonal antibodies and
antisera reactive with specific antigenic determinants (such as on
viruses, cells or other materials), drugs, polynucleotides, nucleic
acids, peptides, cofactors, lectins, sugars, polysaccharides,
cells, cellular membranes, and organelles. Receptors are sometimes
referred to in the art as anti-ligands. As the term receptors is
used herein, no difference in meaning is intended. A "Ligand
Receptor Pair" is formed when two macromolecules have combined
through molecular recognition to form a complex.
[0041] Specific examples of receptors which can be investigated by
this invention include but are not restricted to:
[0042] a) Microorganism receptors: Determination of ligands which
bind to receptors, such as specific transport proteins or enzymes
essential to survival of microorganisms, is useful in a new class
of antibiotics. Of particular value would be antibiotics against
opportunistic fungi, protozoa, and those bacteria resistant to the
antibiotics in current use.
[0043] b) Enzymes: For instance, the binding site of enzymes such
as the enzymes responsible for cleaving neurotransmitters;
determination of ligands which bind to certain receptors to
modulate the action of the enzymes which cleave the different
neurotransmitters is useful in the development of drugs which can
be used in the treatment of disorders of neurotransmission.
[0044] c) Antibodies: For instance, the invention may be useful in
investigating the ligand-binding site on the antibody molecule
which combines with the epitope of an antigen of interest;
determining a sequence that mimics an antigenic epitope may lead to
the development of vaccines of which the immunogen is based on one
or more of such sequences or lead to the development of related
diagnostic agents or compounds useful in therapeutic treatments
such as for autoimmune diseases (e.g., by blocking the binding of
the "self" antibodies).
[0045] d) Nucleic Acids: Sequences of nucleic acids may be
synthesized to establish DNA or RNA binding sequences.
[0046] e) Catalytic Polypeptides: Polymers, preferably
polypeptides, which are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products. Such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, which
functionality is capable of chemically modifying the bound
reactant. Catalytic polypeptides and others are described in, for
example, PCT Publication No. WO 90/05746, WO 90/05749, and WO
90/05785, which are incorporated herein by reference for all
purposes.
[0047] f) Hormone receptors: For instance, the receptors for
insulin and growth hormone. Determination of the ligands which
bind. with high affinity to a receptor is useful in the development
of, for example, an oral replacement of the daily injections which
diabetics must take to relieve the symptoms of diabetes, and in the
other case, a replacement for the scarce human growth hormone which
can only be obtained from cadavers or by recombinant DNA
technology. Other examples are the vasoconstrictive hormone
receptors; determination of those ligands which bind to a receptor
may lead to the development of drugs to control blood pressure.
[0048] g) Opiate receptors: Determination of ligands which bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0049] Substrate or Solid Support: A material having a surface and
which is substantially insoluble in a solution used for coupling of
monomers to a growing polymer chain. Such materials will preferably
take the form of small beads, pellets, disks or other convenient
forms, although other forms may be used. A roughly spherical or
ovoid shape is preferred.
[0050] Basis Set: A group of monomers that is selected for
attachment to a solid substrate directly or indirectly in a given
coupling step. Different basis sets or the same basis sets may be
used from one coupling step to another in a single synthesis.
[0051] Synthetic: Produced by in vitro chemical or enzymatic
synthesis. The synthetic libraries of the present invention may be
contrasted with those in viral or plasmid vectors, for instance,
which may be propagated in bacterial, yeast, or other living
hosts.
[0052] Symbols
[0053] x.sub.i denotes the set of monomer units in reaction round
i.
[0054] x.sub.ij denotes the j'th monomer unit in reaction round i;
x.sub.ij can be a null (.O slashed.).
[0055] S.sub.i refers to the separated products after reaction
round i.
[0056] P.sub.i refers to the pooled products of round i and all
preceding rounds.
[0057] X.sub.ip denotes the pooling of reactants of round i
only
[0058] Reaction Graphs
[0059] A filled circle e denotes a reaction product terminating in
a particular monomer unit x.sub.ij. The set of reaction products
terminating in x.sub.i is shown by a set of circles on the same
horizontal level.
[0060] Filled circles that react with each other are connected by
straight lines. Pooling is shown by lines meeting below in an open
circle.
[0061] A factorable polynomial synthesis is one in which each
monomer unit of a round is joined to each monomer of the preceding
round. In a graph of such a synthesis, each filled circle at one
level is connected to each filled circle of the level above. For
example, the reaction graph corresponding to a three-round
factorable synthesis with
X.sub.1=X.sub.2=X.sub.3={A,T,G,C}
[0062] which yields all 64 trinucleotides, is shown in FIG. 7.
[0063] In contrast, in an irreducible (prime) polynomial synthesis,
at least one line in the graph of the corresponding factorable
polynomial synthesis is missing. In the synthesis of AAT, TGC, TGT,
GTA, GTG, and CCG only, such syntheses are illustrated in FIG.
8.
[0064] II. Overall Description
[0065] FIG. 1 is an overall illustration of one aspect of the
invention. As shown therein, monomers A and B, which form all or
part of a first basis set of monomers, are coupled to substrates 2
in vessels 4a and 4b. The substrates in each of the vessels 4a and
4b are divided. A portion of the substrates from each of vessels 4a
and 4b are mixed in vessel 6, and, divided for a subsequent
coupling step into vessels 6a and 6b. Another fraction of the
monomers from vessels 4a and 4b is not mixed, as indicated by
vessels 10 and 12.
[0066] Thereafter, the substrates are coupled to monomers from a
second basis set C,D, which may or may not be the same as the basis
set A,B. As shown, the monomer C is coupled to the mixed or
"pooled" substrates in vessel 6a, while the monomer D is coupled to
the "pooled" substrates in vessel 6b. A portion of the products of
these reactions may be mixed for later coupling steps, but at least
a portion of the products in vessels 6a and 6b are not mixed.
[0067] The products in vessels 10 and 12 are preferably each
divided for coupling to monomer C as shown in vessels 10b and 12b,
while the substrates in vessels 10a and 12a are used to couple the
monomer D to the growing polymer chain. The products of the
reactions in vessels 10a and 10b are mixed or pooled, and placed in
vessel 20. The products of the reactions in vessels 12a and 12b are
mixed or pooled, and placed in vessel 22.
[0068] The products in vessels 20 and 22 are, thereafter, used to
identify a first monomer in a polymer which is complementary to a
receptor of interest. It is assumed for the sake of illustration
herein that the monomer sequence AC is complementary to the
receptor R. A receptor labeled with, for example, a fluorescent or
radioactive label *, is exposed to the materials in vessels 20 and
22, and unbound receptor is separated from the solid supports.
Binding to the substrates will occur only with the substrates in
vessel 20. Fluorescence is, therefore, observed only in vessel 20.
From this observation, it is possible to conclude that the first
monomer in a complementary receptor is A, since all of the polymers
in vessel 22 contain the first monomer B. Conversely, all of the
polymers in vessel 20 contain the first monomer A.
[0069] The labeled receptor is also exposed to the polymers in
vessels 26a and 26b. In this case, binding of the labelled receptor
will be observed only in vessel 26a. Accordingly, it is possible to
identify the second monomer in a complementary sequence as C, since
none of the polymers in vessel 26b contain the second monomer C,
while all of the polymers in vessel 26a contain the second monomer
C. Therefore, it is possible to conclude that the sequence AC is
complementary to R since binding is observed in vessels 26a and
20.
[0070] FIG. 1b illustrates aspects of a preferred embodiment of the
invention in greater detail with a larger polymer chain. According
to the embodiment shown in FIG. 1b, a basis set of 3 monomers, A,
B, and C is used in each coupling step. The synthesized polymers
are to be three monomers long. It will be recognized by those of
skill in the art that the number of monomers in a basis set and the
number of coupling steps will vary widely from one application to
another. Also, intervening coupling steps of, for example, common
monomer sequences may be used in some embodiments. Therefore, when
a polymer is represented by, for example, the notation "ABC" or
"ABE" herein, it is to be understood that other common monomers may
be added such that ABDC and ABDE are represented by ABC and ABE.
The embodiment shown in FIG. 1b is provided merely as an
illustration of the invention.
[0071] As shown in FIG. 1b, the synthesis takes place on a
plurality of substrates 2. According to a preferred aspect of the
invention, the substrates 2 take the form of beads, such as those
made of glass, resins, plastics, or the like. The term "beads" is
used interchangeably herein with the word "substrate," although it
is to be understood that the beads need not take on a circular or
ovoid shape and can take the form of any suitable substrate. It
will be further understood that the substrates 2 are shown only in
the top portion of FIG. 1b, but the substrates will be present in
each of the reaction products shown in FIG. 1b to the left of the
monomer sequences. In each vessel in FIG. 1b, all of the possible
polymer products are listed. Many "copies" of each sequence will
generally be present.
[0072] According to one embodiment, conventional Merrifield
techniques are used for the synthesis of peptides, such as
described in Atherton et al., "solid Phase Peptide Synthesis," IRL
Press, (1989), previously incorporated herein by reference for all
purposes. Of course other synthesis techniques will be suitable
when different monomers are used. For example, the techniques
described in Gait et al., Oligonucleotide Synthesis, previously
incorporated by reference herein by reference for all purposes,
will be used when the monomers to be added to the growing polymer
chain are nucleotides. These techniques are only exemplary, and
other more advanced techniques will be used in some embodiments
such as those for reversed and cyclic polymer synthesis disclosed
in U.S. application Ser. No. 07/796,727, previously incorporated
herein by reference for all purposes.
[0073] A large number of beads are utilized such that the beads may
be separated into separate reaction vessels in later steps and
still be present in sufficient numbers such that the presence of a
complementary receptor may be detected. As a general rule, it will
be desired to use 10 to 100 or more times the number of
combinatorial possibilities for the synthesis so as to ensure each
member of each set is synthesized. Also, the use of a large number
of beads ensures that pooled reaction products are distributed to
each succeeding reaction vessel when a pooled group of beads is
divided.
[0074] The beads are preferably as small as possible so that the
reaction vessels and other material handling equipment utilized in
the process may also be as small as possible. Preferably, the beads
have a diameter of less than about 1 mm, and preferably less than
about 100 .mu.m, and more preferably less than about 10 .mu.m. In
some embodiments, the synthesis is carried out in solution. In
other embodiments, the synthesis is carried out on solid
substrates, and the resulting polymers are then cleaved from the
substrates before binding with a receptor.
[0075] As shown in FIG. 1b the monomers A, B, and C are coupled to
substrates in three reaction vessels 4a, 4b, and 4c, respectively.
A single substrate is shown in FIG. 1b for purposes of clarity, but
it will be recognized that in each reaction vessel a large number
of beads will be present. Accordingly, a large number of "copies"
of the substrates with the respective monomers coupled thereto are
formed in each of reaction vessels 4a, 4b, and 4c. It will be
recognized that the monomers need not be directly coupled to the
substrate, and in most cases linker molecules will be provided
between the monomers and the substrate, such as those described in
U.S. application Ser. No. 07/624,120, incorporated herein by
reference for all purposes. Also, it should be recognized that the
steps shown in FIG. 1b may be preceded by or followed by other
synthesis steps which may or may not be combinatorial steps using
the techniques described herein.
[0076] Thereafter, a fraction of the products in each of vessels
4a, 4b, and 4c are combined, mixed, and redistributed to each of
reaction vessels 6a, 6b, and 6c. The remaining fraction of the
products in each of vessels 4a, 4b, and 4c is not combined.
Instead, the remaining fraction of the products in reaction vessel
4a is divided and placed in reaction vessels 8a, 8b, and 8c.
Similarly, the remaining fraction of the products in vessel 4b is
divided and placed in vessels 10a, 10b, and 10c. The remaining
fraction of the products in reactant vessel 4c is divided and
placed in reaction vessels 12a, 12b, and 12c.
[0077] The reactants placed in vessels 6a, 6b, and 6c are referred
to herein as "pooled" reactants since they comprise a mixture of
the products resulting from the previous coupling step. The
reactants placed in vessels 8, 10, and 12 by contrast are separate
reactants since they are not mixtures of the products from the
previous coupling steps. According to a preferred embodiment of the
invention, after the reactants in vessels 8, 10, and 12 are
subjected to a separate coupling step, they are subjected only to
pooled coupling steps thereafter. Conversely, in each subsequent
coupling step, the pooled reactants are subjected to a coupling
step, and divided for subsequent separate and pooled coupling
steps..
[0078] Preferably, the reactants are divided such that a greater
fraction of the beads is distributed for pooled synthesis. For
example, in FIG. 9, 4/5 of the beads would go to the first pooled
group 905 while 1/5 would go to the unpooled group 903.
[0079] Thereafter the monomers A, B, and C are coupled to the
growing polymer chain in reaction vessels 8a, 8b, and 8c,
respectively. The resulting polymers then have the monomer sequence
CA, CB, and CC in reaction vessels 8a, 8b, and 8c, respectively.
The products of these reactions are then mixed or pooled in
reaction vessel 9, and the mixture is again divided among reaction
vessels 14a, 14b, and 14c. The monomers A, B, and C are again
coupled to the growing polymer chains in vessels 14a, 14b, and 14c,
respectively. The products of these reactions are again mixed or
pooled and placed in vessel 16a.
[0080] Similarly, the monomers A, B, and C are coupled to the
growing polymer chain in reaction vessels 10a, 10b, and 10c, then
mixed in vessel 18, divided, and placed in reaction vessels 20a,
20b, and 20c. Monomers A, B, and C are coupled to the growing
polymer chain in vessels 20a, 20b, and 20c respectively, mixed, and
placed in vessel 16b. Monomers A, B, and C are also coupled to the
growing polymer chain in reactant vessels 12a, 12b, and 12c
respectively, mixed, and placed in vessel 21. These products are
divided for reaction with monomers A, B, and C in vessels 22a, 22b,
and 22c respectively, mixed, and placed in vessel 16c. A
characteristic feature of the preferred embodiments of the present
invention should be noted in the right half of FIG. 1b.
Specifically, once the products of a reaction are not pooled (such
as in vessels 8, 10, and 12), the products of coupling steps are
always pooled thereafter.
[0081] Referring to the left hand portion of FIG. 1b, the pooled
reactants in vessels 6a, 6b, and 6c are coupled to monomers A, B,
and C respectively, resulting in the products shown in vessels 26a,
26b, and 26c. Since the products in vessels 26a, 26b, and 26c are
derived from a "chain" of pooled reactions, the products are
separated for both pooled and separate reactions. Specifically, a
portion of the substrates in vessels 26a, 26b, and 26c are
combined, mixed, and divided for pooled reactions with monomers A,
B, and C in vessels 28a, 28 b, and 28c respectively. In addition,
the remaining portion of the products in vessels 26a, b, and c are
separately divided and placed in reaction vessels 30a-c, 32a-c, and
34a-c respectively. The materials in vessels 30a, 32a, and 34a are
coupled to monomer A, the materials in vessels 30b, 32b, and 34b
are coupled to monomer B, and the materials in vessels 30c, 32c,
and 34c are coupled to monomer C. Since the products in vessels 30,
32, and 34 result have been preceded by a separate reaction, the
products in vessels 30, 32, and 34 are pooled, or mixed, and placed
in vessels 36a, 36b, and 36c, respectively.
[0082] For reasons that will be discussed further below, the
vessels in group 42 are used to determine the identity of the
monomer in the first position in a polymer that is complementary to
a receptor. The vessels in group 44 are used to determine the
identity of the second monomer in a polymer that is complementary
to a receptor. The vessels in group 46 are used to determine the
identity of the third monomer in a polymer that is complementary to
a receptor.
[0083] For example, assume that a given receptor is complementary
to the monomer sequence ABC, but the sequence of the complementary
polymer is not known ab initio. If the receptor is labelled with an
appropriate label such as fluorescein and placed in each of the
vessels in groups 42, 44, and 46, fluorescence will be detected
only in vessels 16c, 36b, and 28c since the. polymer sequence ABC
appears only in these vessels. Fluorescence may be detected using,
for example, the methods described in Mathies et al., U.S. Pat. No.
4,979,824, incorporated herein in its entirety by reference for all
purposes.
[0084] Since all of the polymers in vessel 16c have monomer A in
the first position, and none of the polymers in vessels 16a or 16b
have monomer A in the first position, it is readily determined that
the monomer in the first position of a complementary polymer is the
monomer A. Similarly, since all of the polymers in vessel 36b have
the monomer B in the second position, it is readily determined that
the monomer B must occupy the second position of a complementary
polymer sequence. Similarly, since all of the polymers in vessel
28c have a C monomer in the third position, the complementary
receptor must have a C in its third position. Therefore, it would
readily be determined that the complementary sequence to the
receptor has the monomer sequence ABC
[0085] As will be seen upon careful examination of the sequences in
the vessel groups 42, 44, 46, ambiguities will generally not arise,
regardless of the monomer sequence which is complementary to the
receptor of interest. As a point of comparison, if the receptor of
interest is complementary to the sequence BBA, fluorescence would
be detected only in vessels 16b, 36b, and 28a. From this
information is becomes clear that the complementary monomer
sequence must be BBA.
[0086] The above embodiment illustrates the synthesis of pooled
groups of polymers by way of separation into separate vessels,
followed by coupling and mixing. It will be recognized that this is
only for convenience of illustration and that in some embodiments
the pooled groups of polymers will be synthesized under controlled
conditions by simultaneous reaction of each of the monomers to be
coupled to the polymers in a single reactor. Further, it will be
recognized that the synthesis steps above will be supplemented in
many embodiments by prior, intermediate, and subsequent coupling
steps, which are not illustrated for ease of illustration.
[0087] The above method may be generally illustrated by way of the
adoption of appropriate nomenclature. For example, let X.sub.i
denote the set of monomer units that become joined to a growing
chain at reaction round i. For example, suppose that
X.sub.i={L,G}X.sub.2={P,Y}X.sub.3={R,A}
[0088] A particular monomer is denoted by x.sub.ij. For
example,
x.sub.3,1=R
[0089] The reaction products S.sub.3 of such a three-round peptide
synthesis is concisely represented by
S.sub.3=X.sub.3X.sub.2X.sub.1
[0090] S.sub.3 is determined by expanding a reaction polynomial as
described in Fodor et al., Science (1991) 251:767-773, incorporated
herein by reference for all purposes.
S.sub.3=(R+A)(P+Y)(L+G)
[0091] and so S.sub.3 consists of 8 tripeptides:
RPL, RYL, RPG, RYG, APL, AYL, APG, and AYG
[0092] S.sub.ij denotes a set of reaction products terminating in
monomer unit X.sub.ij. In the above synthesis, for example,
S.sub.12=G S.sub.21={PL,PG}S.sub.32={APL,AYL,APG,AYG}
[0093] This three-round synthesis can also be represented by a
reaction graph, as shown in FIG. 2. Each reaction product of round
i is depicted by a filled dot on the same horizontal level. Each
dot of round i is joined to each dot of the preceding round and to
each dot of the succeeding round. For example, the dot denoting
S.sub.21, is joined to the dots for S.sub.11 and S.sub.12, and also
to the dots S.sub.31, and S.sub.32. Note that dots on a level are
never connected to each other because, by definition, monomer units
of a round do not combine with one another.
[0094] It is generally assumed that the products of each round are
spatially separate and addressable. Each can then be readily
assayed. However, the number of compounds generated by a
combinatorial synthesis can, after a few rounds, greatly exceed the
number of experimentally available bins or vessels. It is then
advantageous to pool the products of one or more rounds of
synthesis. For example, a five-round synthesis using the basic set
of 20 amino acids yields 20.sup.5 or 3.2.times.10.sup.6
pentapeptides. In contrast, if the products of the first two rounds
are pooled, the subsequent three rounds yield only 8,000 sets of
products. Information is lost in the pooling process, but the
number of products becomes experimentally tractable.
[0095] The above representation of combinatorial synthesis may be
modified to take into account the effect of pooling. Suppose that
products of the first two rounds of the three-round synthesis
mentioned earlier are pooled. The reaction graph for a with pooled
steps is shown in FIG. 3. The pooled products of round i are
denoted by P.sub.i to distinguish them for the separate products
S.sub.i. In a reaction graph, pooling is shown by the convergence
of lines from the S.sub.i that are pooled. P.sub.i is then shown as
an open circle.
[0096] In this example,
P.sub.1={L+G}P.sub.2={PL+PG+YL+YG}
S.sub.3=X.sub.3P.sub.2={RPL+RPG+RYL+RYG,APL+APG+AYL+AYG}
[0097] The plus sign joins products that are present in a mixture.
In contrast, products separated by commas are located in separate
bins and are spatially addressable. In this example, the pooled
products of the second round are located in one bin, whereas the
products after three rounds are located in two bins. One bin
contains the mixture RPL+RPG+RYL+RYG, and the other bin contains
the mixture APL+APG+AYL+AYG.
[0098] This reaction graph can be simplified. Suppose that P.sub.1
was coupled to a equimolar mixture of x.sub.21 and x.sub.22 in a
single bin. If the coupling efficiencies for all species are the
same, the amounts and kinds of products obtained would be the same
as that given by coupling P.sub.1 with x.sub.21 and x.sub.22 in
separate bins and then pooling the products. Thus, pooled products
and pooled reactants are formally equivalent provided that the
reactions occur in a substantially homogeneous solution and all
coupling efficiencies are substantially the same. Hence, an
X.sub.3P.sub.2 synthesis can be most simply represented by the
reaction graph shown in FIG. 4.
[0099] The line joining P.sub.2 to P.sub.1 means that all products
in PI are coupled equally to all reactants X.sub.2, either by (1)
adjusting the concentrations of reactants or (2) driving the
reactions to completion in separate bins, followed by pooling. For
beads or other discrete particles, (2) more often applies so that
each particle expresses only one kind of product.
[0100] By way of comparison, the synthesis of 180 pentapeptides in
Furka et al., "General Method for Rapid Synthesis of Multicomponent
Peptide Mixtures," Int. J. Peptide Protein Res. (1991) 37:487-493,
is represented with the above nomenclature as
S.sub.5=X.sub.5P.sub.4, where X.sub.i={A}, X.sub.2={E,F,K,},
X.sub.3={E,P,K}, X.sub.4={E,F,G,K}, and X.sub.5={E,G,K,L,P}. The
peptide combinatorial library synthesis in Houghten et al.,
"Generation and Use of Synthetic Peptide Combinatorial Libraries
for Basic Research and Drug Discovery," Nature (1991) 354:84-86 is
S.sub.6=X.sub.6X.sub.5P.sub.4, where each X.sub.i is a set of 18
naturally occurring amino acids. The S.sub.6 products are located
in 18.times.18 or 324 bins, each containing a mixture of
18.sup.3=5,832 hexapeptides. The pooled synthesis in Lam et al., "A
new type of synthetic peptide library for identifying
ligand-binding activity," Nature (1991) 354:82-84, is represented
using the above nomenclature as .sub.P5, where each X.sub.i is a
set of 19 naturally-occurring amino acids. P.sub.5 is a mixture of
19.sup.5=2.48.times.10.sup.6 beads, each bearing one kind of
peptide.
[0101] In the pooled syntheses of Houghten, Lam, and Furka, all
products from round 1 to round n are mixed. In Furka's synthesis
(X.sub.5P.sub.4), the first four rounds are pooled. In an
X.sub.3P.sub.2 synthesis, the first two rounds are pooled.
[0102] Representative pooled syntheses techniques according to one
preferred embodiment of the invention herein are shown in FIGS. 5a,
5b, and 5c. The symbol X.sub.ip means that the reactants of round i
have been pooled without pooling the reaction products of previous
rounds. This is achieved by, for example, (1) mixing the reactants
X.sub.i or (2) by reacting each member of X.sub.i with each
reaction product of S.sub.i-1, as shown in FIG. 6 for
X.sub.3pX.sub.2X.sub.1p.
[0103] For pentapeptides made of the naturally occurring 20 amino
acids for example, a family of five pooled syntheses groups
according to the invention herein will be particularly useful:
X.sub.5X.sub.4pX.sub.3pX.sub.2pX.sub.1p
X.sub.5pX.sub.4X.sub.3pX.sub.2pX.s- ub.1p
X.sub.5pX.sub.4pX.sub.3X.sub.2pX.sub.1p
X.sub.5pX.sub.4pX.sub.3pX.su- b.2X.sub.1p
X.sub.5pX.sub.4pX.sub.3pX.sub.2pX.sub.1
[0104] The products of each of these five syntheses product groups
would be located in 20 physically isolated bins. Each bin would
contain a different mixture of 160,000 pentapeptides. As with the
trimer illustrated in FIG. 1b, the identity of the monomers forming
a complementary pentamer would be determined unambiguously by
identifying which of the 20 bins in each of the five syntheses
product groups showed binding to a receptor.
[0105] It is to be recognized that while "bins" are referred to
herein for the sake of simplicity, any of a variety of techniques
may be used for physically separating the peptide or other polymer
mixtures.
[0106] More specifically, a sequence of monomers in a complementary
ligand for a receptor is identified as follows. For example,
consider the family of pooled tripeptide libraries made of the 20
naturally occurring amino acids:
X.sub.3X.sub.2pX.sub.1p X.sub.3pX.sub.2X.sub.1p
X.sub.3pX.sub.2pX.sub.1
[0107] The most potent amino acid at the left position (x.sub.3i)
is revealed by analysis of the 20 bins of X.sub.3X.sub.2pX.sub.1p;
x.sub.2j is determined by analysis of X.sub.3pX.sub.2X.sub.1p; and
x.sub.1k is determined by analysis of X.sub.3pX.sub.2pX.sub.1. The
sequence of the most potent tripeptide is then predicted to be
x.sub.3ix.sub.2jx.sub.1k. Accordingly, each pooled group in the
library reveals the identity of a monomer in a different position
in a complementary polymer.
[0108] It will be recognized that it will not always be desirable
to determine the identity of the entire sequence of monomers in a
polymer that is complementary to a receptor. Instead, it will only
be necessary to determine the identity of selected monomers in a
polymer in some instances. The monomers of interest may be at
intermediate locations on the chain of polymer, and may be
interspersed by other monomers. Accordingly, in a more general
sense, the method herein provides for the synthesis of a library of
polymers. The library is used to identify at least two monomers of
interest in the polymer chain.
[0109] For example, the identity of the x.sub.2j monomer is
determined by analysis of a library of polymers
T-X.sub.2-I-X.sub.1p-T; and the identity of the monomer x.sub.1k is
determined by analysis of a library of polymers
T-X.sub.2p-I-X.sub.1-T, where T indicates terminal groups on the
polymer chain, which may be null groups, and I designates
intermediate groups in the polymer chain, which may also be null
groups.
[0110] The method of making the library used pooled and separate
synthesis steps. The polymers have at least two monomer locations
at which it is desired to determine the identity of monomers which
provide a polymer with a sequence complementary to a receptor. The
library is synthesized such that the products of a pooled synthesis
are separated and subjected to a separate synthesis and a second
pooled synthesis. The products of the separate synthesis are
subjected to a Is series of pooled syntheses, without any further
separate synthesis in preferred embodiments. Conversely, the
products of the second pooled synthesis are divided and subjected
to both a separate syntheses and a third pooled synthesis.
[0111] The synthesis steps result in a library of polymers having
at least first and second subsets. The first subset is used to;
determine the identity of a monomer or monomers at a first location
in the polymer chain which is complementary to a receptor. The
second subset of the library is used to determine the identity of a
monomer or monomers at a second location in the polymer chain which
is complementary to a receptor.
[0112] The method uses summated assays to identify optimal
sequences. The distribution of activities in the mixture assayed
remains unknown. Only the aggregate activity is determined. More
information can be obtained from analyses of beads or other
particles that contain multiple copies of one kind of sequence. The
activity of each bead can be quantitated even though its identity
is unknown.
[0113] Suppose that 2 .mu.m diameter beads are used for pooled
syntheses. Some pertinent properties of typical beads are:
[0114] Volume=4.2 .mu.m.sup.3
[0115] Surface area=12.6 .mu.m.sup.2
[0116] Number of target sites=1.3.times.10.sup.5
[0117] (assuming 1 per 100 mm.sup.2)
[0118] Number of beads per cm.sup.3=2.4.times.10.sup.11
[0119] Fluorescence measurements of beads flowing rapidly through a
laser beam are made using techniques such as those in U.S. Pat. No.
4,979,824, previously incorporated herein by reference for all
purposes, which provide exemplary methods for determining the
distribution of activities in a pooled synthesis.
[0120] Assume a light beam diameter of 2 .mu.m is used for
detection of fluorescein labeled beads, at a flow rate of 20 cm/s.
The transit time of a bead through the beam is then 10 .mu.s. The
emission rate from a single chromophore can be as high as
10.sup.8s.sup.-1. If 10% of the target sites are occupied, this
corresponds to an emission rate of about 10.sup.12s.sup.-1, or
10.sup.7 emitted photons in 10 .mu.s, which would be easily
detected. If 10% of the sample volume is occupied by beads, an
average of one bead would pass through the beam every 0.1 ms. Thus,
10.sup.4 beads could be analyzed per second. A library of
3.2.times.10.sup.4, beads (each bearing a different pentapeptide)
could be analyzed in about 6 minutes.
[0121] Alternatively, the beads way be analyzed by spreading them
on a surface. For example, 3.2.times.10.sup.6 beads would occupy
1.28.times.10.sup.7 .mu.m.sup.2 if packed together in a square
array. In 1.28 cm.sup.2, these beads would occupy 10% of the
surface area. Smaller beads, say 0.2 .mu.m.sup.2, would give a
sufficient fluorescence signal. The advantage of smaller beads is
that higher bead densities could be used, leading to a marked
reduction in the time needed for analysis.
[0122] The fluorescence pulse height distribution emerging from
either analysis would reveal whether there are many or few optimal
sequences contained within the sample of beads. In the simplest
case, a single bright bead is seen in just one bin of a pooled
synthesis. The identity of the best sequence then comes directly
from analysis of each pooled synthesis of the family.
[0123] In other cases, there is a distribution of intensities
within several sets of beads. As a general rule, positioned
libraries where binding is exhibited in multiple bins indicates
that a particular position plays a less significant role in
binding. In some embodiments, positions where ambiguity are
detected are further evaluated through use of the VLSIPS.TM.
technique. The VLSIPS.TM. arrays will vary only those positions
wherein the monomer has not been determined unambiguously. The
present invention is used, therefore, to reduce the number of
polymers which will be screened with VLSIPS.TM. in some
embodiments.
[0124] In still other cases, polymer mixtures synthesized in
multiple bins are cleaved from their respective beads and then
assayed for activity. The freed polymers are then able to interact
with receptors in various orientations. The activity of such
polymers can be assayed by various well-known techniques such as
ELISA.
[0125] FIG. 9 provides an alternative description of the invention.
As shown therein, at step 901 a collection of substrates is
subjected to pooled and separate coupling steps, resulting in
pooled and separate products 905 and 903, respectively. In
comparison with the embodiment. shown in FIG. 1b, products 903 are
analogous to the products shown in vessels 8, 10, and 12, and
products 905 are analogous to the products in vessel 6. The
collection of substrate products 903 are then subjected to pooled
coupling steps 903, 905, 907, and 909, i.e., the subsequent
coupling steps to the separate reactants are only pooled coupling
steps. Accordingly, the identity of the monomer in the first
position of a polymer complementary to a receptor is determined by
evaluation of the products 907.
[0126] Conversely, the pooled products 905 are divided and
subjected to pooled and separate coupling steps 909, resulting in
pooled and separate products 907 and 913, respectively. As with the
separate products 903, the separate products 913 are subjected only
to pooled coupling steps thereafter, resulting in pooled products
915 and 917. The products 917 are used to determine the monomer in
a second position in a polymer complementary to a receptor of
interest.
[0127] In the same manner, the pooled products 907 are divided and
subjected to pooled and separate coupling steps 919, resulting in
pooled and separate products 923 and 921. The separate products 923
are subjected only to a pooled reaction thereafter, the products
925 being used to determine the monomer in a third position in a
polymer complementary to a receptor of interest. The pooled
products 921 are divided and subjected to pooled and separate
reactions 927, resulting in pooled and separate products 929 and
931. The products 907, 917, 925, 931, and 929 are used to identify
complementary receptors. In the preferred embodiment, the pooled
products 927 are first used to determine if any polymers of
interest are present. The separate products 931 are used to
determine the identity of a monomer in a fourth position of a
polymer complementary to a receptor.
[0128] As shown in FIG. 9, pooled products that have not been
subjected to prior separate reactions are divided and subjected to
pooled and separate reactions according to the invention herein.
Conversely, products which result from a prior separate coupling
step are only subjected to pooled coupling steps.
[0129] In an alternative embodiment depicted in FIGS. 10a-10c, a
recursive retrosynthesis is employed to screen a diverse set of
polymers. Unlike the recursive retrosynthesis method of Houghten et
al. described supra, this method identifies the sequence of a
"best" polymer by identifying a collection of polymers ("library")
containing the bead giving the strongest signal. Houghton et al.,
in contrast, identify the entire library, rather than the single
polymer (bead), having the strongest signal. Thus, the technique of
Houghton et al. may identify an incorrect monomer in the sequence
of interest because of the library containing that monomer gave the
strongest signal, while the "best" polymer is located in a
different library. The recursive retrosynthesis embodiment of this
invention overcomes this difficulty of Houghton et al.'s by
identifying the individual polymer giving the strongest signal.
[0130] Referring to FIGS. 10a-10c, an example of this process is
described for the set of all pentamers formed from a basis set of
50 monomers. As shown in FIG. 10a, the complete collection of
quadramers is synthesized on a number of beads (e.g.,
3.times.10.sup.8 beads) by four cycles of alternately dividing,
reacting, and pooling the beads. The pool of quadramers is then
divided into 50 bins, each of which is reacted with a different
member of the basis set to give 50 bins of pentamers, each
containing only those pentamers terminating in a specified monomer.
In the notation used herein, this collection of polymers is
represented by X.sub.1PX.sub.2PX.sub.4PX.sub.5(1-50). The
individual beads in each bin are then assayed to identify the
single best bead (i.e., the bead providing the strongest signal on
binding with the receptor of interest). This may be accomplished in
about eight hours by FACS as described above, for example. Having
determined the bin containing the bead providing the strongest
signal, the identity of the monomer in the fifth position is known.
In the example of FIG. 10, that monomer is D.
[0131] Next, the complete collection of trimers is formed as before
(from e.g., 6.times.10.sup.6 beads) by cycles of dividing,
reacting, and pooling the beads as shown in FIG. 10b.
Alternatively, the library of trimers could be set aside after the
third cycle of the previous step (during formation of the complete
library of pentamers). At this point, the pooled beads are divided
into 50 bins, each of which is reacted with a different member of
the basis set. The quadramers in each bin are then reacted with D
to produce a collection of beads represented by
X.sub.1PX.sub.2PX.sub.3PX.sub.4(1-50)D. The beads in each bin are
then assayed to again identify the bead giving the strongest
signal. The bin from which that bead was taken identified the
monomer at the next position: A in this case. The above process is
repeated to produce X.sub.1PX.sub.2PX.sub.3(1-50)AD and identify
the monomer at the next position as shown in FIG. 10c. In this
example, the next monomer is identified as Q. The final two
monomers of the sequence can be identified in the same fashion.
However, it may be more efficient to simply screen the remaining
2,500 possible pentamers via a VLSIPS.TM. technique.
[0132] In general, for a polymer of length N synthesized from a
basis set of n monomers, the terminal monomer may be identified by
the following procedure. First, a pooled library of substrates is
formed such that each substrate has a different polymer
synthesized. The pooled library includes a collection of polymers
represented by X.sub.1pX.sub.2p . . . X.sub.(N-1)p. The library is
divided into n separate bins, each of which is then reacted with a
different monomer to form a library X.sub.1pX.sub.2p . . .
X.sub.(N-1)pX.sub.N(1-n). Finally, a receptor is exposed to the
substrate in each of the a separate bins to identify the bin
containing the polymer which binds the receptor most strongly. This
bin provides the identify the monomer in the X.sub.N position. The
penultimate monomer is identified by a similar procedure from
X.sub.1pX.sub.2p . . . X.sub.(N-1)(1-n)R, where R is the terminal
monomer previously identified. Each succeeding monomer can be
identified in the same manner. In this example, the two basis sets
used to identify the monomers at positions N and N-1 each contained
n members. It will be appreciated that the monomer basis sets used
to identify the monomer at each position on the polymer may
independent of the other basis sets.
[0133] A combinatorial synthesis chamber for conducting the
synthesis, pooling, and dividing steps employed in each cycle of
this invention is illustrated in FIG. 11a-11c. Individual chambers
200, each containing an amount of packed beads 203, are aligned in
close proximity to one another to form a two-dimensional array. The
reaction chambers 200 are mounted on a base 207 via passages 211. A
filter 213 is provided at the base of each coupling chamber 200 to
prevent the beads from leaving the coupling chambers 200 when
solutions are drained through passages 211. In synthesis mode,
coupling solutions are introduced through passages 205 while cover
201 is in a closed position (as shown in FIG. 11a). The contents of
each chamber 200 are prevented from contacting adjacent chambers by
cover 201. Passages 205 and 211 are controlled by valves or other
control mechanisms not shown.
[0134] After the coupling reactions have proceeded to the desired
extent, the coupling solutions are drained from the synthesis
chamber through the passages 211. Subsequent washing steps may be
necessary before pooling and redistribution. In such washing steps,
washing solutions are introduced through passages 205. while cover
201 is held in the closed position. After sufficient time has
elapsed, the washing solution is drained through passages 211.
Multiple washing steps may be performed as necessary to remove
unused coupling solutions from chambers 200. During the reacting
and washing steps, the beads in the reaction chambers may be
agitated by rotating or shaking the entire synthesis chamber.
[0135] As shown in FIG. 11b pooling and dividing of the beads is
accomplished by sliding cover 201 away from base 207 to form a
mixing chamber 215. The previously packed beads 203 are then
agitated in a fluid such that they mix within mixing chamber 215.
Ultimately, when the agitation is stopped, the beads will settle
randomly into various chambers 200. At that point, the suspension
solution can be drained through passages 211, and cover 201 can be
lowered to rest on the tops of chamber 200 as shown in FIG. 11a.
During the pooling and dividing stages, the valves in passages 205
and 211 are closed. A sealing means 209 is provided to prevent
beads or fluid from leaving the synthesis chamber.
[0136] FIG. 11c shows a top view of a two-dimensional array of
reaction chambers 200 mounted on base 207. The chambers shown are
arranged in a 7.times.7 array of 49 chambers.
[0137] FIG. 12 illustrates a library of polymers which will be
useful in accordance with the invention herein. As shown therein,
the polymers have a number of monomer positions, designated by
p.sub.i. The monomers have at least two positions of interest,
p.sub.1 and p.sub.2. p.sub.1 and p.sub.2 may in some embodiments be
separated by various intermediate monomers or groups, and may also
have various terminal groups attached thereto. The monomers are
placed in a number of physically isolated bins or vessels 1002. The
bins or vessels 1002 may in fact be attached, such as in a
microtiter plate, or the bins/vessels may be distinct containers
such as test tubes, microtiter trays, or the like.
[0138] A first bin 1002a contains polymers with a first monomer
M.sub.1 in the first position p.sub.1 in each of the polymers
therein. However, the polymer molecules in the first bin have a
variety of different monomers such as M.sub.1, M.sub.2, and M.sub.3
in a second position p.sub.2. In the second bin 1002b a second
monomer M.sub.2 is in the first position p.sub.1 in each of the
polymers therein, while different monomers such as M.sub.1,
M.sub.2, and M.sub.3 are in the second position p.sub.2. In the
third bin 1002c a third monomer M.sub.3 is in the first position
p.sub.1 in each of the polymers therein, while different monomers
such as M.sub.1, M.sub.2, and M.sub.3 are in the second position
p.sub.2. The first, second, and third bins comprise all or part of
a collection of bins . . . X.sub.1 . . . X.sub.2p . . .
[0139] Conversely, fourth bin 1002d contains polymers with a first
monomer M.sub.1 in the second position p.sub.2 in each of the
polymer molecules therein. The polymer molecules in the first bin
have a variety of different monomers such as M.sub.1, M.sub.2, and
M.sub.3 in their first position p.sub.1. In the fifth bin 1002e a
second monomer M.sub.2 is in the second position p.sub.2 in each of
the polymers therein, while different monomers such as M.sub.1,
M.sub.2, and M.sub.3 are in the first position p.sub.1. In the
sixth bin 1002f a third monomer M.sub.3 is in the second position
p.sub.2 in each of the polymers therein, while different monomers
such as M.sub.1, M.sub.2, and M.sub.3 are in the first position
p.sub.1. The fourth, fifth, and sixth bins comprise all or part of
a collection of bins . . . X.sub.1p . . . X.sub.2 . . .
[0140] In screening studies, the bins 1002a, 1002b, and 1002c are
used to determine the identity of the monomer in position 1 of a
polymer that is complementary to a receptor of interest. The bins
1002d, 1002e, and 1002f are used to determine the identity of the
monomer in position 2 of a polymer that is complementary to a
receptor of interest.
[0141] It will be recognized that the polymers which are screened
according to the above methods can be of widely varying length and
composition. For example, in preferred embodiments, the polymer
molecules are preferably greater than 3 monomer units long,
preferably greater than 5 monomer units long, more preferably
greater than 10 monomer units long, and more preferably more than
20 monomer units long. Although a simplified library is shown in
FIG. 12, it will be recognized that in most embodiments, the
library will include additional polymer bins so as to identify the
monomers at more than 3 positions, preferably more than 5
positions, more preferably more than 10 positions, and more
preferably more than 20 positions in a complementary polymer to a
receptor.
[0142] III. Polynomial Factoring Applied to Screening
[0143] In some embodiments a population of all possible polymers of
length n are synthesized. If a receptor is found to bind with one
of the polymers in the mixture, a second synthesis is conducted in
which the polymers are "factored," i.e., two bins are formed, each
having half of the population synthesized initially. It is then
determined which of the two bins shows binding to the receptor, the
bin which exhibits binding being referred to as a "target group."
Yet another synthesis is conducted in which two bins are created,
each with half of the population of the target group in the earlier
bin. The process is repeated until the sequence of the polymer or
polymers that show binding to the receptors is determined.
[0144] More specifically, the invention provides for the synthesis
of a population: 1 P = n X i n X j
i=1j=1
[0145] This solution is factored as: 2 P = i X i [ j n / 2 X j + n
/ 2 n X j ] = P 1 + P 2 where: P 1 = i = 1 n X i j = 1 n / 2 X j ;
and P 2 = i = 1 n X i n / 21 n X j
[0146] If P.sub.1 generates a "hit," P.sub.1 is factored. If
P.sub.2 generates a "hit," P.sub.2 is factored. Each synthesis
requires only half the number of polymers made in the prior
step.
[0147] IV. Conclusion
[0148] The above description is illustrative and not restrictive.
Many variations of the invention will become apparent to those of
skill in the art upon review of this disclosure. Merely by way of
example while the invention is illustrated primarily with regard to
the synthesis of oligonucleotides and peptides, the invention will
also find utility in conjunction with the synthesis and analysis of
a wide variety of additional polymers. The scope of the invention
should, therefore, be determined not with reference to the above
description, but instead should be determined with reference to the
appended claims along with their full scope of equivalents.
Sequence CWU 1
1
2 1 6 PRT Artificial Sequence epitope 1 Asp Val Pro Asp Tyr Ala 1 5
2 4 PRT Artificial Sequence epitope 2 Phe Leu Arg Phe 1
* * * * *