`Lerner et al.
`
`[54] ENCODED COMBINATORIAL CHEMICAL
`LIBRARIES
`
`[75]
`
`Inventors: Richard Lerner, La Jolla; Kim Janda,
`San Diego, both of Calif.; Sydney
`Brenner, Cambridge, England
`
`[73] Assignee: The Scripps Research Institute, La
`Jolla, Calif.
`
`[21] Appl. No.: 860,445
`
`[22] Filed:
`
`Mar. 30, 1992
`
`Int. Cl.6
`[51]
`.......••.•••••••...•••••••••. C12Q 1/68; GOlN 33/53
`[52] U.S. Cl ................................................. 435/6; 435/7.94
`[58] Field of Search ......................... 435/6, 7.94; 935/77,
`935/78; 530/350; 436/501
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,748,111
`4,965,188
`5,082,780
`5,141,813
`
`5/1988 Dattagupta et al. . ....................... 435/7
`1011990 Mullis et al ................................ 435/6
`111992 Warren et al ........................... 435/191
`8/1992 Nelson .................................... 428/402
`
`OTHER PUBLICATIONS
`
`Devlin, et al., Science, 249: 404--406 (1990).
`Nelson, et a!., Nucleic Acids Research, 17: 7179-7195
`(1989).
`Nelson, et al., Nucleic Acids Research, 20: 6253-6259
`(1992).
`"Sales Literatuare" from Clontech Laboratories, Inc., p. 3
`(1994).
`Geysen, et al., "Use of Peptide Synthesis of Probe Viral
`Antigens for Epitopes to a Resolution of a Single Amino
`Acid," Proc. Natl. Acad. Sci. U.S.A., 81: 3998-4002 (1984).
`
`111111111111111111111111111111111111111111 111111111111111111111 11111111 1111
`US005573905A
`[lll Patent Number:
`[45] Date of Patent:
`
`5,573,905
`Nov. 12, 1996
`
`Maeji, et al., "Simultaneous Multiple Synthesis of Peptide(cid:173)
`-Carrier Conjugates," J. Immunol. Met., 146: 83-90 (1992).
`Fodor, et al., "Light-Directed, Spatially Addressable Paral(cid:173)
`lel Chemical Synthesis," Science, 251: 767-775 (1991).
`Lam, et iii., "A New Type of Synthetic Peptide Library for
`Identifying Ligand-Binding Activity," Nature, 354: 82-84
`(1991).
`Houghton, et al., "Generation and Use of Synthetic Peptide
`Combinatorial Libraries for Basic Research and Drug Dis(cid:173)
`covery," Nature, 354: 84-86 (1991).
`Cwirla, et al., "Peptides on Phage: A Vast Library ofPeptides
`for Idenifying Ligands," Proc. Nat!. Acad. Sci. U.S.A., 87:
`6378-6382 (1990).
`Scott, et al., "Searching for Peptide Ligands with an Epitope
`Library," Science, 249: 386-390 (1990).
`Devlin, et al., "Random Peptide Libraries: A Source of
`Specific Protein Binding Molecules," Science, 249: 404--406
`(1990).
`
`Primary Examiner-W. Gary Jones
`Assistant Examiner-Eggerton Campbell
`Attorney, Agent, or Firm-Donald G. Lewis
`
`[57]
`
`ABSTRACT
`
`The present invention describes an encoded combinatorial
`chemical library comprised of a plurality of bifunctional
`molecules having both a chemical polymer and an identifier
`oligonucleotide sequence that defines the structure of the
`chemical polymer. Also described are the bifunctional mol(cid:173)
`ecules of the library, and methods of using the library to
`identify chemical structures within the library that bind to
`biologically active molecules in preselected binding inter(cid:173)
`actions.
`
`5 Claims, 2 Drawing Sheets
`
`
`
`Page 1 of 25
`
`ILMN EXHIBIT 1003
`
`
`
`U.S. Patent
`
`Nov. 12, 1996
`
`Sheet 1 of 2
`
`5,573,905
`
`Apa I
`Sty I
`)GGGCCCTATTCTTAG 3 1
`5 1 AGCTACTTCCCAAGG[coding sequence
`3 1 TCGATGAAGGGTTCC[anticoding strand)CCCGGGATAAGAATC 5 1
`
`I Step 1 Cleavage by
`
`~
`
`Sty I & Apa I
`
`5'
`3'
`
`AGCTACTTCC
`TCGATGAAGGGTTC
`
`CAAGG(coding sequence
`)GGGCC
`Q[anticoding strand)Q
`
`CTATTCTTAG
`CCGGGATAAGAATC
`
`t Step 2 Biotinylation
`
`3 I
`5'
`
`5'
`3'
`
`)GGGCC
`CAAGG(coding sequence
`AGCTACTTCCC
`TCGATGAAGGGTTC BBCQ[anticoding strand]Q
`
`CTATTCTTAG
`CCGGGATAAGAATC
`
`3'
`5'
`
`FIGURE 1
`
`
`
`Page 2 of 25
`
`
`
`U.S. Patent
`
`Nov. 12, 1996
`
`Sheet 2 of 2
`
`5,573,905.
`
`Pl - LINK
`
`.J;
`
`Step 1
`
`CACATG-Pl-LINK-gly
`
`ACGGTA-Pl-LINK-met
`
`1 Step 2
`
`CACATGCACATG-Pl-LINK-gly.gly
`CACATGACGGTA-Pl-LINK-met.gly
`
`ACGGTACACATG-Pl-LINK-gly.met
`ACGGTAACGGTA-Pl-LINK-met.met
`
`t Step 3
`
`P2CACATGCACATGCACATGP1-LINK-gly.gly.gly
`P2CACATGCACATGACGGTAP1-LINK-met.gly.gly
`P2CACATGACGGTACACATGPl-LINK-gly.met.gly
`P2CACATGACGGTAACGGTAP1-LINK-met.met.gly
`
`P2ACGGTACACATGCACATGP1-LINK-gly.gly.met
`P2ACGGTACACATGACGGTAP1-LINK-met.gly.met
`P2ACGGTAACGGTACACATGP1-LINK-gly.met.met
`P2ACGGTAACGGTAACGGTAP1-LINK-met.met.met
`
`Pl = GGGCCCTATTCTTAG
`P2 = AGCTACTTCCCAAGG
`
`FIGURE 2
`
`
`
`Page 3 of 25
`
`
`
`5,573,905
`
`1
`ENCODED COMBINATORIAL CHEMICAL
`LIBRARIES
`
`TECHNICAL FIELD
`
`The present invention relates to encoded chemical librar(cid:173)
`ies that contain repertoires of chemical structures defining a
`diversity of biological structures, and methods for using the
`libraries.
`
`BACKGROUND
`
`IO
`
`2
`searching a dictionary; the peptide is decoded by construc(cid:173)
`tion using a series of sieves or buckets and this makes the
`search logarithmic.
`A very powerful biological method has recently been
`5 described in which the library of peptides is presented on the
`surface of a bacteriophage such that each phage has an
`individual peptide and contains the DNA sequence specify(cid:173)
`ing it. The library is made by synthesizing a repertoire of
`random oligonucleotides to generate all combinations, fol-
`lowed by their insertion into a phage vector. Each of the
`sequences is cloned in one phage and the relevant peptide
`can be selected by finding those that bind to the particular
`target. The phages recovered in this way can be amplified
`and the selection repeated. The sequence of the peptide is
`15 decoded by sequencing the DNA. See for example Cwirla et
`a!., Proc. Natl.Acad. Sci. USA, 87:6378-6382 (1990); Scott
`et a!., Science, 249:386-390 (1990); and Devlin et a!.,
`Science, 249:404-406 (1990).
`Another "genetic" method has been described where the
`libraries are the synthetic oligonucleotides themselves
`wherein active oligonucleotide molecules are selected by
`binding to an acceptor and are then amplified by the poly(cid:173)
`merase chain reaction (PCR). PCR allows serial enrichment
`and the structure of the active molecules is then decoded by
`DNA sequencing on clones generated from the PCR prod(cid:173)
`ucts. The repertoire is limited to nucleotides and the natural
`pyrimidine and purine bases or those modifications that
`preserve specific Watson-Crick pairing and can be copied by
`polymerases.
`The main advantages of the genetic methods reside in the
`capacity for cloning and amplification of DNA sequences,
`which allows enrichment by serial selection and provides a
`facile method for decoding the structure of active molecules.
`However, the genetic repertoires are restricted to nucleotides
`and peptides composed of natural amino acids and a more
`extensive chemical repertoire is required to populate the
`entire universe of binding sites. In contrast, chemical meth(cid:173)
`ods can provide limitless repertoires but they lack the
`capacity for serial enrichment and there are difficulties in
`discovering the structures of selected active molecules.
`
`BRIEF SUMMARY OF THE INVENTION
`
`45
`
`The present invention provides a way of combining the
`virtues of both of the chemical and genetic methods sum(cid:173)
`marized above through the construction of encoded combi(cid:173)
`natorial chemical libraries, in which each chemical sequence
`is labelled by an appended "genetic" tag, itself constructed
`50 by chemical synthesis, to provide a "retrogenetic" way of
`specifying each chemical structure.
`In outline, two alternating parallel combinatorial synthe(cid:173)
`ses are performed so that the genetic tag is chemically linked
`to the chemical structure being synthesized; in each case, the
`55 addition of one of the particular chemical units to the
`structure is followed by the addition of an oligonucleotide
`sequence, which is defined to "code" for that chemical unit,
`ie., to function as an identifier for the structure of the
`chemical unit. The library is built up by the repetition of this
`process after pooling and division.
`Active molecules are selected from the library so pro(cid:173)
`duced by binding to a preselected biological molecule of
`interest. Thereafter, the identity of the active molecule is
`determined by reading the genetic tag, i.e., the identifier
`oligonucleotide sequence. In one embodiment, amplified
`copies of their retrogenetic tags can be obtained by the
`polymerase chain reaction.
`
`30
`
`35
`
`There is an increasing need to find new molecules which
`can effectively modulate a wide range of biological pro(cid:173)
`cesses, for applications in medicine and agriculture. A
`standard way for searching for novel bioactive chemicals is
`to screen collections of natural materials, such as fermen(cid:173)
`tation broths or plant extracts, or libraries of synthesized
`molecules using assays which can range in complexity from
`simple binding reactions to elaborate physiological prepa(cid:173)
`rations. The screens often only provide leads which then 20
`require further improvement either by empirical methods or
`by chemical design. The process it time-consuming and
`costly but it is unlikely to be totally replaced by rational
`methods even when they are based on detailed knowledge of
`the chemical structure of the target molecules. Thus, what 25
`we might call "irrational drug design"-the process of
`selecting the right molecules from large ensembles or rep(cid:173)
`ertoires-requires continual improvement both in the gen(cid:173)
`eration of repertoires and in the methods of selection.
`Recently there have been several developments in using
`peptides or nucleotides to provide libraries of compounds
`for lead discovery. The methods were originally developed
`to speed up the determination of epitopes recognized by
`monoclonal antibodies. For example, the standard serial
`process of stepwise search of synthetic peptides now encom(cid:173)
`passes a variety of highly sophisticated methods in which
`large arrays of peptides are synthesized in parallel and
`screened with acceptor molecules labelled with fluorescent
`or other reporter groups. The sequence of any effective 40
`peptide can be decoded from its address in the array. See for
`example Geysen et a!., Proc. Natl.Acad. Sci.USA,
`81:3998-4002 (1984); Maeji et a!., J.Immunol.Met.,
`146:83-90 (1992); and Fodor eta!., Science, 251: 767-775
`(1991).
`In another approach, Lam et. a!., Nature, 354:82-84
`(1991) describes combinatorial libraries of pep tides that are
`synthesized on resin beads such that each resin bead con(cid:173)
`tains about 20 pmoles of the same peptide. The beads are
`screened with labelled acceptor molecules and those with
`bound acceptor are searched for by visual inspection, physi(cid:173)
`cally removed, and the peptide identified by direct sequence
`analysis. In principle, this method could be used with other
`chemical entities but it requires sensitive methods for
`sequence determination.
`A different method of solving the problem of identifica(cid:173)
`tion in a combinatorial peptide library is used by Houghten
`eta!., Nature, 354:84-86 (1991). For hexapeptides of the 20
`natural amino acids, 400 separate libraries are synthesized,
`each with the first two amino acids fixed and the remaining 60
`four positions occupied by all possible combinations. An
`assay, based on competition for binding or other activity, is
`then used to find the library with an active peptide. Then
`twenty new libraries are synthesized and assayed to deter(cid:173)
`mine the effective amino acid in the third position, and the 65
`process is reiterated in this fashion until the active hexapep(cid:173)
`tide is defined. This is analogous to the method used in
`
`
`
`Page 4 of 25
`
`
`
`5,573,905
`
`4
`sequences shown in FIG. 1 are listed in the Sequence
`Listing, SEQ ID NOs 15-22.
`FIG. 2 illustrates the process of producing a library of
`bifunctional molecules according to the method described in
`Example 9. The nucleotide base sequences shown in FIG. 1
`are listed in the Sequence Listing, SEQ ID NOs 15-22.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`30
`
`35
`
`40
`
`3
`The strands of the amplified copies with the appropriate
`polarity can then be used to enrich for a subset of the library
`by hybridization with the matching tags and the process can
`then be repeated on this subset. Thus serial enrichment is
`achieved by a process of purification exploiting linkage to a 5
`nucleotide sequence which can be amplified. Finally, the
`structure of the chemical entities are decoded by cloning and
`sequencing the products of the PCR reaction.
`The present invention therefore provides a novel method
`for identifying a chemical structure having a preselected
`binding activity through the use of a library of bifunctional
`molecules that provides a rich source of chemical diversity.
`The library is used to identify chemical structures (structural
`motifs) that interact with preselected biological molecules.
`Thus, in one embodiment, the invention contemplates a
`bifunctional molecule according to the formula A-B-C,
`where A is a chemical moiety, B is a linker molecule
`operatively linked to A and C, and C is an identifier
`oligonucleotide comprising a sequence of nucleotides that
`identifies the structure of chemical moiety A.
`In another embodiment, the invention contemplates a
`library comprising a plurality of species of bifunctional
`molecules, thereby forming a repertoire of chemical diver(cid:173)
`sity.
`Another embodiment contemplates a method for identi(cid:173)
`fying a chemical structure that participates in a preselected
`binding interaction with a biologically active molecule,
`where the chemical structure is present in the library of
`bifunctional molecules according to this invention. The
`method comprises the steps of:
`a) admixing in solution the library of bifunctional mol(cid:173)
`ecules with the biologically active molecule under
`binding conditions for a time period sufficient to form
`a binding reaction complex;
`b) isolating the complex formed in step (a); and
`c) determining the nucleotide sequence of the polymer
`identifier oligonucleotide in the isolated complex and
`thereby identifying the chemical structure that partici(cid:173)
`pated in the preselected binding interaction.
`The invention also contemplates a method for preparing a
`library according to this invention comprising the steps of:
`a) providing a linker molecule B having termini A' and C'
`according to the formula A'-B-C' that is adapted for
`reaction with a chemical precursor unit X' at termini A'
`and with a nucleotide precursor Z' at termini C';
`b) conducting syntheses by adding chemical precursor
`unit X' to termini A' of said linker and adding precursor
`unit identifier oligonucleotide Z' to termini C' of said
`linker, to form a composition containing bifunctional
`molecules having the structure Xn-B-Zn;
`c) repeating step (b) on one or more aliquots of the
`composition to produce aliquots that contain a product
`containing a bifunctional molecule;
`d) combining the aliquots produced in step (c) to form an
`admixture of bifunctional molecules, thereby forming
`said library.
`
`10 A. Encoded Combinatorial Chemical Libraries
`An encoded combinatorial chemical library is a compo(cid:173)
`sition comprising a plurality of species of bifunctional
`molecules that each define a different chemical structure and
`that each contain a unique identifier oligonucleotide whose
`15 nucleotide sequence defines the corresponding chemical
`structure.
`1. Bifunctional Molecules
`A bifunctional molecule is the basic unit in a library of ·
`this invention, and combines the elements of a polymer
`20 comprised of a series of chemical building blocks to form a
`chemical moiety in the library, and a code for identifying the
`structure of the chemical moiety.
`Thus, a bifunctional molecule can be represented by the
`formula A-B-C, where A is a chemical moiety, B is a linker
`25 molecule operatively linked to A and C, and Cis an identifier
`oligonucleotide comprising a sequence of nucleotides that
`identifies the structure of chemical moiety A.
`a. Chemical Polymers
`A chemical moiety in a bifunctional molecule of this
`invention is represented by A in the above formula A-B-C
`and is a polymer comprising a linear series of chemical units
`represented by the formula (Xn)a, wherein X is a single
`chemical unit in polymer A and n is a position identifier for
`X in polymer A. n has the value of l+i where i is an integer
`from 0 to I 0, such that when n is 1, X is located most
`proximal to the linker (B).
`Although the length of the polymer can vary, defined by
`a, practical library size limitations arise if there is a large
`alphabet size as discussed further herein. Typically, a is an
`integer from 4 to 50.
`A chemical moiety (polymer A) can be any of a variety of
`polymeric structures, depending on the choice of classes of
`chemical diversity to be represented in a library of this
`invention. Polymer A can be any monomeric chemical unit
`that can be coupled and extended in polymeric form. For
`example, polymer A can be a polypeptide, oligosaccharide,
`glycolipid, lipid, proteoglycan, glycopeptide, sulfonamide,
`nucleoprotein, conjugated peptide (i.e., having prosthetic
`groups), polymer containing enzyme substrates, including
`transition state analogues, and the like biochemical poly(cid:173)
`mers. Exemplary is the polypeptide-based library described
`herein.
`Where the library is comprised of peptide polymers, the
`chemical unit X can be selected to form a region of a natural
`55 protein or can be a non-natural polypeptide, can be com(cid:173)
`prised of natural D-amino acids, or can be comprised of
`non-natural amino acids or mixtures of natural and non(cid:173)
`natural amino acids. The non-natural combinations provide
`for the identification of useful and unique structural motifs
`involved in biological interactions.
`Non-natural amino acids include modified amino acids
`and L-amino acids, stereoisomer of D-amino acids.
`The amino acid residues described herein are preferred to
`be in the "L" isomeric form. NH2 refers to the free amino
`65 group present at the amino terminus of a polypeptide.
`COOH refers to the free carboxy group present at the
`carboxy terminus of a polypeptide. In keeping with standard
`
`45
`
`50
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`In the drawings, forming a portion of this disclosure:
`FIG. 1 illustrates a scheme for the restriction endonu(cid:173)
`clease cleavage of a PCR amplification product derived from
`a bifunctional molecule of this invention (Step 1), and the
`subsequent addition of biotin to the cleaved PCR product
`(Step 2). The unique coding and non-coding nucleotide base
`
`60
`
`
`
`Page 5 of 25
`
`
`
`TABLE OF CORRESPONDENCE
`
`SYMBOL
`
`!-Letter
`
`3-Letter
`
`AMINO ACID
`
`5,573,905
`
`10
`
`20
`
`25
`
`45
`
`5
`polypeptide nomenclature, J. Biol. Chern., 243:3552-59
`(1969) and adopted at 37 C.P.R. §1.822(b) (2)), abbrevia(cid:173)
`tions for amino acid residues are shown in the following
`Table of Correspondence:
`
`6
`and the unit identifier oligonucleotide Z at position n.
`The length of a unit identifier oligonucleotide can vary
`depending on the complexity of the library, the number of
`different chemical units to be uniquely identified, and other
`5 considerations relating to requirements for uniqueness of
`oligonucleotides such as hybridization and polymerase
`chain reaction fidelity. A typical length can be from about 2
`to about 10 nucleotides, although nothing is to preclude a
`unit identifier from being longer.
`Insofar as adenosine (A), guanosine (G), thymidine (T)
`and cytosine (C) represent the typical choices of nucleotides
`for inclusion in a unit identifier oligonucleotide, A, G, T and
`C form a representative "alphabet" used to "spell" out a unit
`identifier oligonucleotide's sequence. Other nucleotides or
`15 nucleotide analogs can be utilized in addition to or in place
`of the above fournucleotides, so long as they have the ability
`to form Watson-Crick pairs and be replicated by DNA
`polymerases in a PCR reaction. However, the nucleotides A,
`G, T and Care preferred.
`For the design of the code in the identifier oligonucle-
`otide, it is essential to chose a coding representation such
`that no significant part of the oligonucleotide sequence can
`occur in another unrelated combination by chance or other(cid:173)
`wise during the manipulations of a bifunctional molecule in
`the library.
`For example, consider a library where Z is a trinucleotide
`whose sequence defines a unique chemical unit X. Because
`the methods of this invention provide for all combinations
`The phrase "amino acid residue" is broadly defined to
`and permutations of an alphabet of chemical units, it is
`include the amino acids listed in the Table of Correspon(cid:173)
`30 possible for two different unit identifier oligonucleotide
`dence and modified and unusual amino acids, such as those
`sequences to have closely related sequences that differ by
`listed in 37 C.P.R. §1.822(b) (4), and incorporated herein by
`only a frame shift and therefore are not easily distinguish(cid:173)
`reference.
`able by hybridization or sequencing unless the frame is clear.
`The polymer defined by chemical moiety A can therefor
`Other sources of misreading of a unit identifier oligo-
`contain any polymer backbone modifications that provide
`35 nucleotide can arise. For example, mismatch in DNA
`increased chemical diversity. In building of a polypeptide
`hybridization, transcription errors during a primer extension
`system as exemplary, a variety of modifications are contem(cid:173)
`reaction to-amplify or sequence the identifier oligonucle(cid:173)
`plated,
`including
`the
`following backbone structures:
`otide, and the like errors can occur during a manipulation of
`-NHN(R)CO-, -NHB(R)CO-, -NHC(RR')CO-,
`a bifunctional molecule.
`-NHC(=CHR)CO-,
`-NHC6H4CO-,
`The invention contemplates a variety of means to reduce
`-NHCH2CHRCO-, -NHCHRCH2CO-, and lactam 40
`the possibility of error in reading the identifier oligonucle(cid:173)
`structures.
`otide, such as to use longer nucleotide lengths for a unit
`In addition, amide bond modifications are contemplated
`identifier nucleotide sequence as to reduce the similarity
`including -COCH2- , -COS-, -CONR, -COO-,
`between unit
`identifier nucleotide sequences. Typical
`-CSNH-, -CH2NH-, -CH2CH2- , -CH2S-,
`lengths depend on the size of the alphabet of chemical units.
`-CH2SO-,
`-CH2S02- ,
`-CH(CH3)S-,
`A representative system useful for eliminating read errors
`-CH=CH-,
`-NHCO-,
`-NHCONH-,
`due to frame shift or mutation is a code developed as a
`-CONHO-, and -C(=CH)CH2- .
`theoretical alternative to the genetic code and is known as
`b. Polymer Identifier Oligonucleotide
`the commaless genetic code.
`An identifier oligonucleotide in a bifunctional molecule of
`Where the chemical units are amino acids, a convenient
`this invention is represented by C in the above formula 50
`unit identifier nucleotide sequence is the well known genetic
`A-B-C and is an oligonucleotide having a sequence repre(cid:173)
`code using triplet codons. The invention need not be limited
`sented by the formula (Zn)a, wherein Z is a unit identifier
`by the translation afforded between the triplet codon of the
`nucleotide sequence within oligonucleotide C that identifies
`genetic code and the natural amino acids; other systems of
`the chemical unit X at position n. n has the value of 1 +i
`where i is an integer from 0 to 10, such that when n is 1, z 55 correspondence can be assigned.
`A typical and exemplary unit identifier nucleotide
`is located most proximal to the linker (B). a is an integer as
`sequence is based on the commaless code having a length of
`described previously to connote the number of chemical unit
`six nucleotides (hexanucleotide) per chemical unit.
`identifiers in the oligonucleotide.
`Preferably, an identifier oligonucleotide has at least 15
`For example, a bifunctional molecule can be represented
`60 nucleotides in the tag (coding) region for effective hybrid(cid:173)
`by the formula:
`ization. In addition, considerations of the complexity of the
`library, the size of the alphabet of chemical units, and the
`length of the polymer length of the chemical moiety all
`contribute to length of the identifier oligonucleotide as
`65 discussed in more detail herein.
`In a preferred embodiment, an identifier oligonucleotide
`C has a nucleotide sequence according to the formula
`
`y
`G
`F
`M
`A
`s
`I
`L
`T
`v
`p
`K
`H
`Q
`E
`w
`R
`D
`N
`c
`
`Tyr
`Gly
`Phe
`Met
`Ala
`Ser
`lie
`Leu
`Thr
`Val
`Pro
`Lys
`His
`Gin
`Glu
`Trp
`Arg
`Asp
`Asn
`Cys
`
`tyrosine
`glycine
`phenylalanine
`methionine
`alanine
`serine
`isoleucine
`leucine
`threonine
`valine
`proline
`lysine
`histidine
`glutamine
`glutamic acid
`tryptophan
`arginine
`aspartic acid
`asparagine
`cysteine
`
`x.x,x,x,-B-z, z,z,z •.
`
`In this example, the sequence of oligonucleotides Z 1, Z2 , Z3
`and z4 identifies the structure of chemical units XI, Xz, x3
`and X4 , respectively. Thus, there is a correspondence in the
`identifier sequence between a chemical unit X at position n
`
`
`
`Page 6 of 25
`
`
`
`5,573,905
`
`10
`
`7
`Pl-(Z,)a-P2, where Pl and P2 arc nucleotide sequences that
`provide polymerase chain reaction (PCR) primer binding
`sites adapted to amplify the polymer identifier oligonucle(cid:173)
`otide. The requirements for PCR primer binding sites are
`generally well known in the art, but are designed to allow a 5
`PCR amplification product (a PCR-amplified duplex DNA
`fragment) to be formed that contains the polymer identifier
`oligonucleotide sequences.
`The presence of the two PCR primer binding sites, P 1 and
`P2, flanking the identifier oligonucleotide sequence (Zn)a
`provides a means to produce a PCR-amplified duplex DNA
`fragment derived from the bifunctional molecule using PCR.
`This design is useful to allow the amplification of the tag
`sequence present on a particular bifunctional molecule for
`cloning and sequencing purposes in the process of reading 15
`the identifier code to determine the structure of the chemical
`moiety in the bifunctional molecule.
`More preferred is a bifunctional molecule where one or
`both of the nucleotide sequences PI and P2 are designed to
`contain a means for removing the PCR primer binding sites 20
`from the identifier oligonucleotide sequences. Removal of
`the flanking PI and P2 sequences is desirable so that their
`sequences do not contribute to a subsequent hybridization
`reaction. Preferred means for removing the PCR primer
`binding sites from a PCR amplification product is in the
`form of a restriction endonuclease site within the PCR(cid:173)
`amplified duplex DNA fragment.
`Restriction endonucleases are well known in the art and
`are enzymes that recognize specific lengths of duplex DNA
`and cleave the DNA in a sequence-specific manner.
`Preferably, the restriction endonuclease sites should be
`positioned proximal to (Z,)a relative to the PCR primer
`binding sites to maximize the amount of PI and P2 that is
`removed upon treating a bifunctional molecule to the spe(cid:173)
`cific restriction endonuclease. More preferably, PI and P2
`each are adapted to form a restriction endonuclease site in
`the resulting PCR-amplified duplex DNA, and the two
`restriction sites, when cleaved by the restriction endonu(cid:173)
`clease, form non-overlapping cohesive termini to facilitate
`subsequent manipulations.
`Particularly preferred are restriction sites that when
`cleaved provide overhanging termini adapted for termini(cid:173)
`specific modifications such as incorporation of a biotinylated
`nucleotide (e.g., biotinyl deoxy-UTP) to facilitate subse(cid:173)
`quent manipulations.
`The above described preferred embodiments in an iden(cid:173)
`tifier oligonucleotide are summarized in a specific embodi(cid:173)
`ment shown in FIG. 1.
`In FIG. 1, a PCR-amplified duplex DNA is shown that is
`derived from an identifier oligonucleotide described in the
`Examples. The (Z,) sequence is illustrated in the brackets as
`the coding sequence and its complementary strand of the
`duplex is indicated in the brackets as the anticoding strand.
`The PI and P2 sequences arc shown in detail with a Sty I
`restriction endonuclease site defined by the PI sequence 55
`located 5' to the bracket and an Apy I restriction endonu(cid:173)
`clease site defined by the P2 sequence located 3' to the
`bracket.
`Step 1 illustrates the cleavage of the PCR-amplified
`duplex DNA by the enzymes Sty I and Apa I to form a
`modified identifier sequence with cohesive termini. Step 2
`illustrates the specific biotinylation of the anticoding strand
`at the Sty I site, whereby the incorporation of biotinylated
`UTP is indicated by a B.
`The presence of non-overlapping cohesive termini after
`Step 1 in FIG. 1 allows the specific and directional cloning
`of the restriction-digested PCR-amplified fragment into an
`
`8
`appropriate vector, such as a sequencing vector. In addition,
`the Sty I was designed into PI because the resulting over(cid:173)
`hang is a substrate for a filling-in reaction with dCTP and
`biotinyl-dUTP (BTP) using DNA polymerase Klenow frag(cid:173)
`ment. The other restriction site, Apa I, was selected to not
`provide substrate for the above biotinylation, so that only the
`anticoding strand can be biotinylated.
`Once biotinylated, the duplex fragment can be bound to
`immobilized avidin and the duplex can be denatured to
`release the coding sequence containing the identifier nucle(cid:173)
`otide sequence, thereby providing purified anticoding strand
`that is useful as a hybridization reagent for selection of
`related coding strands as described further herein.
`c. Linker Molecules
`A linker molecule in a bifunctional molecule of this
`invention is represented by B in the above formula A-B-C
`and can be any molecule that performs the function of
`operatively linking the chemical moiety to the identifier
`oligonucleotide.
`Preferably, a linker molecule has a means for attaching to
`a solid support, thereby facilitating synthesis of the bifunc(cid:173)
`tional molecule in the solid phase. In addition, attachment to
`a solid support provides certain features in practicing the
`screening methods with a library of bifunctional molecules
`25 as described herein. Particularly preferred are linker mol(cid:173)
`ecules in which the means for attaching to a solid support is
`reversible, namely, that the linker can be separated from the
`solid support.
`A linker molecule can vary in structure and length, and
`30 provide at least two features: (1) operative linkage to chemi(cid:173)
`cal moiety A, and (2) operative linkage to identifier oligo(cid:173)
`nucleotide C. As the nature of chemical linkages is diverse,
`any of a variety of chemistries may be utilized to effect the
`indicated operative linkages to A and to C, as the nature of
`the linkage is not considered an essential feature of this
`invention. The size of the linker in terms of the length
`between A and C can vary widely, but for the purposes of the
`invention, need not exceed a length sufficient to provide the
`linkage functions indicated. Thus, a chain length of from at
`least one to about 20 atoms is preferred.
`A preferred linker molecule is described in Example 3
`herein that contains the added, preferred, element of a
`reversible means for attachment to a solid support. That is,
`the bifunctional molecule is removable from the solid sup-
`45 port after synthesis.
`Solid supports for chemical synthesis are generally well
`known. Particularly preferred are the synthetic resins used in
`oligonucleotide and in polypeptide synthesis that are avail(cid:173)
`able from a variety of commercial sources including Glen
`50 Research (Herndon, Va.), Bachem Biosciences, (Philadel(cid:173)
`phia, Pa.), and Applied Biosystems (Foster City, Calif.).
`Most preferred are teflon supports such as that described in
`Example 2.
`2. Libraries
`A library of this invention is a repertoire of chemical
`diversity comprising a plurality of species of bifunctional
`molecules according to the present invention. The plurality
`of species in a library defines a family of chemical diversity
`whose species each have a different chemical moiety. Thus
`the library can define a family of peptides, lipids, oligosac(cid:173)
`charides or any of the other classes of chemical polymers
`recited previously.
`The number of different species in a library represents the
`complexity of a library and is defined by the polymer length
`65 of the chemical moiety, and by the size of the chemical unit
`alphabet that can be used to build the chemical unit polymer.
`The number of different species referred to by the phrase
`
`35
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`40
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`60
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`"plurality of species" in a libr