`Lerner et al.
`
`111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US006060596A
`[11] Patent Number:
`[45] Date of Patent:
`
`6,060,596
`May 9, 2000
`
`[54] ENCODED COMBINATORIAL CHEMICAL
`LIBRARIES
`
`[75]
`
`Inventors: Richard Lerner, La Jolla; Kim Janda,
`San Diego, both of Calif.; Sydney
`Brenner, Edwards Passage, United
`Kingdom
`
`[73] Assignee: The Scripps Research Institute, La
`Jolla, Calif.
`
`[21] Appl. No.: 09/033,743
`
`[22] Filed:
`
`Mar. 3, 1998
`
`Related U.S. Application Data
`
`[62] Division of application No. 08/665,511, Jun. 18, 1996, Pat.
`No. 5,723,598, which is a division of application No.
`07/860,445, Mar. 30, 1992, Pat. No. 5,573,905.
`Int. Cl? ..................................................... C07H 21/00
`[51]
`[52] U.S. Cl. ............................................................. 536/25.3
`[58] Field of Search .................................. 536/25.3, 24.2;
`436/518, 536; 435/6
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,748,111
`4,923,901
`4,965,188
`5,082,780
`5,141,813
`
`5/1988 Dattagupta et a!. ........................ 435/6
`5/1990 Koester et a!. ........................... 521!53
`10/1990 Mullis et a!. ............................... 435/6
`1!1992 Warren et a!. .......................... 435/191
`8/1992 Nelson .................................... 428/402
`
`FOREIGN PATENT DOCUMENTS
`
`0 323 152
`
`5/1989 European Pat. Off ..
`
`01HER PUBLICATIONS
`
`Clontech Laboratories, Inc., Sales Literature: 2-3 (1994).
`Cwirla, et al., "Peptides on phage: A vast library of pep tides
`for identifying ligands", Proc. Natl. Acad. Sci. USA 87:
`6378-6382 (1990).
`Devlin, et al., "Random Peptide Libraries: A Source of
`Specific Protein Binding Molecules", Science 249: 404--406
`(1990).
`
`Fodor, et al., "Light-Directed, Spatially Addressable Paral(cid:173)
`lel Chemical Synthesis", Science 251: 767-775 (1991).
`Geysen, et al., "Use of peptide synthesis to probe viral
`antigens for epitopes to a resolution of a single amino acid",
`Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1984).
`Houghton, et al., "Generation and use of synthetic peptide
`combinatorial libraries for basic research and drug discov(cid:173)
`ery", Nature 354: 84-86 (1991).
`Lam, et al., "A new type of synthetic peptide library for
`identifying ligand-binding activity", Nature 354: 82-84
`(1991).
`Maeji, et al., "Simultaneous multiple synthesis of peptide(cid:173)
`carrier conjugates", J. Immunol. Met. 146: 83-90 (1992).
`Nelson, et al., "Anew and versatile reagent for incorporating
`multiple primary amines into synthetic oligonucleotides"
`Nucleic Acids Research 17: 7179-7195 (1989).
`Nelson, et al., "Oligonucleotide labeling methods 3. Direct
`labeling of oligonucleotides employing a novel, non(cid:173)
`nucleosidic, 2-aminobutyl-1, 3-propanediol backbone",
`Nucleic Acids Research 20: 6253-6259 (1992).
`Scott, et al., "Searching for Peptide Ligands with an Epitope
`Library", Science 249: 386-390 (1990).
`Hays, et al., "High-Yield Synthesis of Oligoribonucleotides
`Using g_-Nitrobenzyl Protection of 2'-Hydroxyls", Tetrahe(cid:173)
`dron Letters 26: 2407-2410 (1985).
`Hampel et al. Nucleic Acids Research. vol. 18, No. 2, pp.
`299-304, 1990.
`Alberts et al., Molecular Biology of the Cell, p. 343. Garland
`Pubishing, Inc. New York, 1983.
`
`Primary Examiner-Remy Yucel
`Attorney, Agent, or Firm-Thomas E. Northrup
`ABSTRACT
`
`[57]
`
`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.
`
`17 Claims, 2 Drawing Sheets
`
`
`
`Page 1 of 25
`
`ILMN EXHIBIT 1001
`
`
`
`Apa I
`Sty I
`]GGGCCCTATTCTTAG 3 1
`5 1 AGCTACTTCCCAAGG(coding sequence
`3 1 TCGATGAAGGGTTCC(anticoding strand]CCCGGGATAAGAATC 5 1
`
`Step 1 Cleavage by
`Sty I & Apa I
`
`5' AGCTACTTCC
`3' TCGATGAAGGGTTC
`
`]GGGCC
`CAAGG(coding sequence
`C(anticoding strand]C
`
`CTATTCTTAG 3'
`CCGGGATAAGAATC 5 1
`
`]GGGCC
`CAAGG(coding sequence
`5' AGCTACTTCCC
`3' TCGATGAAGGGTTC BBCC(anticoding strand]C
`
`CTATTCTTAG 3'
`CCGGGATAAGAATC 5 1
`
`Step 2 Biotynylation
`
`FIG. 1
`
`d •
`\Jl
`•
`~
`~ ......
`~ = ......
`
`~
`~
`'-<
`~~
`
`N c c c
`
`'JJ. =(cid:173)~
`~ .....
`'"""' 0 ......,
`
`N
`
`0\
`
`.... = 0\ =
`
`....
`Ul
`\C
`0\
`
`
`
`Page 2 of 25
`
`
`
`U.S. Patent
`
`May 9, 2000
`
`Sheet 2 of 2
`
`6,060,596
`
`Pl - LINK t step 1
`
`CACATG-Pl-LINK-gly
`
`ACGGTA-Pl-LINK-met
`
`+ Step 2
`
`CACATGCACATG-Pl-LINK-gly.gly
`CACATGACGGTA-Pl-LINK-met.gly
`
`ACGGTACACATG-Pl-LINK-gly.met
`ACGGTAACGGTA-Pl-LINK-met.met
`
`+ Step 3
`
`P2CACATGCACATGCACATGP1-LINK-gly.gly.gly
`P2CACATGCACATGACGGTAP1-LINK-met.gly.gly
`P2CACATGACGGTACACATGP1-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.rnet.met
`
`Pl = GGGCCCTATTCTTAG
`P2 = AGCTACTTCCCAAGG
`
`FIG. 2
`
`
`
`Page 3 of 25
`
`
`
`6,060,596
`
`1
`ENCODED COMBINATORIAL CHEMICAL
`LIBRARIES
`
`This application is a divisional of Ser. No. 08/665,511,
`filed Jun. 18, 1996, now U.S. Pat. No. 5,723,598, which is
`a divisional of Ser. No. 07/860,445, filed Mar. 30, 1992, now
`U.S. Pat. No. 5,573,905.
`
`DESCRIPTION
`
`2
`twenty new libraries are synthesized and assayed to deter(cid:173)
`mine the effective amino acid in the third position, and the
`process is reiterated in this fashion until the active hexapep(cid:173)
`tide is defined. This is analogous to the method used in
`5 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
`described in which the library of peptides is presented on the
`10 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(cid:173)
`lowed by their insertion into a phage vector. Each of the
`15 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
`decoded by sequencing the DNA See for example Cwirla et
`20 al., Proc. Natl. Acad. Sci. USA, 87:6378-6382 (1990); Scott
`et al., Science, 249:386-390 (1990); and Devlin et al.,
`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
`40 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
`45 discovering the structures of selected active molecules.
`BRIEF SUMMARY OF THE INVENTION
`
`The present invention provides a way of combining the
`virtues of both of the chemical and genetic methods sum-
`50 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
`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
`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
`
`1. 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.
`2. Background
`There is an increasing need to find new molecules which
`can effectively modulate a wide range of biological
`processes, 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 prep a(cid:173)
`rations. The screens often only provide leads which then 25
`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 30
`we might call "irrational drug design" -the process of
`selecting the right molecules from large ensembles or
`repertoires-requires continual improvement both in the
`generation of repertoires and in the methods of selection.
`Recently there have been several developments in using 35
`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
`peptide can be decoded from its address in the array. See for
`example Geysen et al., Proc. Natl. Acad. Sci. USA,
`81:3998-4002 (1984); Maeji et al., J. Immunol. Met.,
`146:83-90 (1992); and Fodor et al., Science, 251: 767-775
`(1991).
`In another approach, Lam et. al., Nature, 354:82-84
`(1991) describes combinatorial libraries of peptides 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- 55
`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- 60
`tion in a combinatorial peptide library is used by Houghten
`et al., 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
`four positions occupied by all possible combinations. An 65
`assay, based on competition for binding or other activity, is
`then used to find the library with an active peptide. Then
`
`
`
`Page 4 of 25
`
`
`
`6,060,596
`
`4
`subsequent addition of biotin to the cleaved PCR product
`(Step 2). The unique coding and non-coding nucleotide base
`sequences shown in FIG. 1 are listed in the Sequence
`Listing, SEQ 1D 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
`
`20
`
`3
`oligonucleotide sequence. In one embodiment, amplified
`copies of their retrogenetic tags can be obtained by the
`polymerase chain reaction.
`The strands of the amplified copies with the appropriate
`polarity can then be used to enrich for a subset of the library 5
`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
`nucleotide sequence which can be amplified. Finally, the
`structure of the chemical entities are decoded by cloning and 10
`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. 15
`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.
`
`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
`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
`comprised of a series of chemical building blocks to form a
`chemical moiety in the library, and a code for identifying the
`25 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 molecule operatively linked to A and C, and C is an
`identifier oligonucleotide comprising a sequence of nucle-
`30 otides 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)m
`35 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 1 + i
`where i is an integer from 0 to 10, 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
`40 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
`protein or can be a non-natural polypeptide, can be com(cid:173)
`prised of natural D-amino acids, or can be comprised of
`60 non-natural amino acids or mixtures of natural and non-
`natural amino acids.
`The non-natural combinations provide for the identifica(cid:173)
`tion of useful and unique structural motifs involved in
`biological interactions.
`Non-natural amino acids include modified amino acids
`and L-amina acids, stereoisomer of D-amino acids. The
`amino acid residues described herein are preferred to be in
`
`45
`
`50
`
`55
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`In the drawings, forming a portion of this disclosure:
`FIG. 1 illustrates a scheme for the restriction endonu- 65
`clease cleavage of a PCR amplification product derived from
`a bifunctional molecule of this invention (Step 1 ), and the
`
`
`
`Page 5 of 25
`
`
`
`6,060,596
`
`6
`
`5
`the "L" isomeric form. NH2 refers to the free amino 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 polypeptide
`nomenclature, J. Bioi. Chern., 243:3552-59 (1969) and 5
`adopted at 37 C.P.R. §1.822(b)(2)), abbreviations for amino
`acid residues are shown in the following Table of Corre(cid:173)
`spondence:
`
`In this example, the sequence of oligonucleotides Zl, Z2 , Z3
`and Z4 identifies the structure of chemical units X1 , X2 X3
`and X4 , respectively. Thus, there is a correspondence in the
`identifier sequence between a chemical unit X at position n
`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
`10 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
`20 nucleotide analogs can be utilized in addition to or in place
`of the above four nucleotides, so long as they have the ability
`to form Watson-Crick pairs and be replicated by DNA
`polymerases in a PCR reaction. However, the nucleotidesA,
`G, T and C are preferred.
`For the design of the code in the identifier
`oligonucleotide, it is essential to chose a coding represen(cid:173)
`tation such that no significant part of the oligonucleotide
`sequence can occur in another unrelated combination by
`chance or otherwise during the manipulations of a bifunc-
`30 tional 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
`and permutations of an alphabet of chemical units, it is
`35 possible for two different unit identifier oligonucleotide
`sequences to have closely related sequences that differ by
`only a frame shift and therefore are not easily distinguish(cid:173)
`able by hybridization or sequencing unless the frame is clear.
`Other sources of misreading of a unit identifier oligo-
`40 nucleotide can arise. For example, mismatch in DNA
`hybridization, transcription errors during a primer extension
`reaction to amplify or sequence the identifier
`oligonucleotide, and the like errors can occur during a
`manipulation of a bifunctional molecule.
`The invention contemplates a variety of means to reduce
`the possibility of error in reading the identifier
`oligonucleotide, such as to use longer nucleotide lengths for
`a unit identifier nucleotide sequence as to reduce the simi(cid:173)
`larity between unit identifier nucleotide sequences. Typical
`50 lengths depend on the size of the alphabet of chemical units.
`A representative system useful for eliminating read errors
`due to frame shift or mutation is a code developed as a
`theoretical alternative to the genetic code and is known as
`the commaless genetic code.
`Where the chemical units are amino acids, a convenient
`unit identifier nucleotide sequence is the well known genetic
`code using triplet codons. The invention need not be limited
`by the translation afforded between the triplet codon of the
`genetic code and the natural amino acids; other systems of
`60 correspondence can be assigned.
`A typical and exemplary unit identifier nucleotide
`sequence is based on the commaless code having a length of
`six nucleotides (hexanucleotide) per chemical unit.
`Preferably, an identifier oligonucleotide has at least 15
`65 nucleotides in the tag (coding) region for effective hybrid(cid:173)
`ization. In addition, considerations of the complexity of the
`library, the size of the alphabet of chemical units, and the
`
`TABLE OF CORRESPONDENCE
`
`SYMBOL
`
`1-Letter
`
`3-Letter
`
`AMINO ACID
`
`y
`G
`F
`M
`A
`s
`
`L
`T
`v
`p
`K
`H
`Q
`E
`w
`R
`D
`N
`c
`
`Tyr
`Gly
`Phe
`Met
`Ala
`Ser
`Ile
`Leu
`Thr
`Val
`Pro
`Lys
`His
`Gln
`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
`
`15
`
`25
`
`The phrase "amino acid residue" is broadly defined to
`include the amino acids listed in the Table of Correspon(cid:173)
`dence and modified and unusual amino acids, such as those
`listed in 37 C.P.R. §1.822(b)(4), and incorporated herein by
`reference.
`The polymer defined by chemical moiety A can therefor
`contain any polymer backbone modifications that provide
`increased chemical diversity. In building of a polypeptide
`system as exemplary, a variety of modifications are
`contemplated, including the following backbone structures:
`-NHN(R)CO-, -NHB(R)CO-, -NHC(RR')CO-,
`-NHC 6 H 4 CO-, 45
`-NHC(=CHR)CO-,
`-NHCH2 CHRCO-, -NHCHRCH2 CO-, and lactam
`structures.
`In addition, amide bond modifications are contemplated
`including -COCH2- , -COS-, -CONR, -COO-,
`-CSNH-, -CH 2 NH-, -CH2 CH 2 - , -CH 2 S-,
`-CH 2 SO-, -CH 2 S0 2 - , -CH(CH 3 )S-,
`-CH=CH-,
`-NHCO-,
`-NHCONH-,
`-CONHO-, and -C =CH2)CH2- .
`b. Polymer Identifier Oligonucleotide
`An identifier oligonucleotide in a bifunctional molecule of
`this invention is represented by C in the above formula
`A-B-C and is an oligonucleotide having a sequence
`represented by the formula (Zn)m wherein Z is a unit
`identifier nucleotide sequence within oligonucleotide C that
`identifies 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 is located most proximal to the linker (B). a is an
`integer as described previously to connote the number of
`chemical unit identifiers in the oligonucleotide.
`For example, a bifunctional molecule can be represented
`by the formula:
`
`55
`
`
`
`Page 6 of 25
`
`
`
`6,060,596
`
`7
`length of the polymer length of the chemical moiety all
`contribute to length of the identifier oligonucleotide as
`discussed in more detail herein.
`In a preferred embodiment, an identifier oligonucleotide
`C has a nucleotide sequence according to the formula
`P1---(Zn)a -P2, where P1 and P2 are nucleotide sequences
`that provide polymerase chain reaction (PCR) primer bind(cid:173)
`ing sites adapted to amplify the polymer identifier oligo(cid:173)
`nucleotide. The requirements for PCR primer binding sites
`are generally well known in the art, but are designed to allow
`a 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, P1 and
`P2, flanking the identifier oligonucleotide sequence (ZnL
`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 20
`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 P1 and P2 are designed to
`contain a means for removing the PCR primer binding sites 25
`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 (Zn)a relative to the PCR primer
`binding sites to maximize the amount of P1 and P2 that is
`removed upon treating a bifunctional molecule to the spe(cid:173)
`cific restriction endonuclease. More preferably, P1 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
`endonuclease, 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 (ZJ 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 P1 and P2 sequences are shown in detail with a Sty I
`restriction endonuclease site defined by the P1 sequence 60
`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
`
`8
`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
`5 of the restriction-digested PCR-amplified fragment into an
`appropriate vector, such as a sequencing vector. In addition,
`the Sty I was designed into Pi because the resulting overhang
`is a substrate for a filling-in reaction with dCTP and biotinyl(cid:173)
`dUTP (BTP) using DNA polymerase Klenow fragment. The
`10 other restriction site, Apa I, was selected to not provide
`substrate for the above biotinylation, so that only the anti(cid:173)
`coding strand can be biotinylated.
`Once biotinylated, the duplex fragment can be bound to
`immobilized avidin and the duplex can be denatured to
`15 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
`30 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
`35 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
`40 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
`45 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-
`50 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
`55 Research (Herndo