I
`
`..
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`ENCODED COMBINATORIAL CHEMICAL LIBRARIES
`
`Description
`
`Technical Field
`The present invention relates to encoded chemical
`libraries that contain repertoires of chemical
`structures defining a diversity of biological
`structures, and methods for using the libraries.
`
`5
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`
`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
`
`.
`
`15
`
`agriculture. A standard way for searching for novel
`
`bioactive chemicals is to screen collections of
`natural materials, such as fermentation broths or
`plant extracts, or libraries of synthesized molecules
`using assays which can range in complexity from simple
`binding reactions to elaborate physiological
`preparations. The screens often only provide leads
`
`20
`
`which then require further improvement either by
`Dempirical methods or by chemical design. The process
`!it
`time-consuming and costly but it is unlikely to be
`25
`totally replaced by rational methods even when they
`are based on detailed knowledge of the chemical
`structure of the target molecules. Thus, what 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 peptides or nucleotides to provide libraries of
`compounds for lead discovery. The methods were
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`ILMN EXHIBIT 1002
`
`
`
`..
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`ENCODED COMBINATORIAL CHEMICAL LIBRARIES
`
`Description
`
`Technical Field
`The present invention relates to encoded chemical
`libraries that contain repertoires of chemical
`structures defining a diversity of biological
`structures, and methods for using the libraries.
`
`5
`
`10
`
`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
`
`.
`
`15
`
`agriculture. A standard way for searching for novel
`
`bioactive chemicals is to screen collections of
`natural materials, such as fermentation broths or
`plant extracts, or libraries of synthesized molecules
`using assays which can range in complexity from simple
`binding reactions to elaborate physiological
`preparations. The screens often only provide leads
`
`20
`
`which then require further improvement either by
`Dempirical methods or by chemical design. The process
`!it
`time-consuming and costly but it is unlikely to be
`25
`totally replaced by rational methods even when they
`are based on detailed knowledge of the chemical
`structure of the target molecules. Thus, what 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 peptides or nucleotides to provide libraries of
`compounds for lead discovery. The methods were
`
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`Page 1 of 74
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`ILMN EXHIBIT 1002
`
`
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`- 2
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`.. r.
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`-2-
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`originally developed to speed up the determination of
`epitopes recognized by monoclonal antibbdies. For
`
`example, the standard serial process of stepwise
`search of synthetic peptides now encompasses 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 contains about 20 pmoles of the same
`peptide. The beads are screened with labelled
`molecules and those with bound acceptor are
`searched for by visual inspection, physically 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
`identification 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
`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 determine the effective
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`amino acid in the third position, and the process is
`reiterated in this fashion until the active
`hexapeptide is defined. This is analogous to the
`method used in searching' a dictionary; the peptide is
`decoded by construction 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 surface of a bacteriophage such that
`each phage has an individual peptide and contains the
`DNA sequence specifying it. The library is Made by
`synthesizing a repertoire of random oligonucleotides
`to generate all combinations, followed by their
`insertion into a phage vector. Each of the equences
`is cloned in one phage and the relevant peptide can be
`selected by finding those that bind to the p rticular
`target. The phages recovered in this way can be
`amplified and the selection repeated. The s&quence of
`the peptide is decoded by sequencing the DNA. See for
`
`example Cwirla et 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
`
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`03(1990)
`
`Another "genetic" method has been described where
`
`25
`
`the libraries are the synthetic oligonucleotides
`themselves wherein active oligonucleotide molecules
`
`are selected by binding to an acceptor and are then
`
`amplified by the polymerase chain reaction (PCR). PCR
`
`allows serial enrichment and the structure of the
`
`30
`
`active molecules is then decoded by DNA sequencing on
`
`clones generated from the PCR products. The
`
`repertoire is limited to nucleotides and the natural
`
`pyrimidine and purine bases or those modifications
`
`35
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`that preserve specific Watson-Crick pairing and can be
`copied by polymerases.
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`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
`methods 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
`The present invention provides a way of combining
`the virtues of both of the chemical and genetic
`methods summarized above through the construction of
`encoded combinatorial 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 syntheses 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
`
`)
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`,T ; ; )
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`produced 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.
`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
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`process of purification exploiting linkage tb a
`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
`Ssource 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
`
`25
`
`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 diversity.
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`Page 5 of 74
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`
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`Another embodiment contemplates a method for
`identifying.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:
`
`admixing in solution the library of
`a)
`bifunctional molecules with the biologically active
`molecule under binding conditions for a time period
`sufficient to form a binding reaction complek;
`
`b)
`
`isolating the complex formed in step
`
`(a); and
`
`determining the nucleotide sequence of
`c)
`the polymer identifier oligonucleotide in the isolated.
`complex and thereby identifying the chemical structure
`that participated in the preselected binding
`
`interaction.
`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;
`
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`combining the aliquots produced in step
`d)
`(c) to form an admixture of bifunctional molecules,
`thereby forming said library.
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`Brid' Description of the Drawings
`In the drawings, forming. a portion of this
`disclosure:
`Figure 1 illustrates a scheme for the restriction
`endonuclease 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
`Figure 2 illustrates t process of producing a
`library of bifunctional molecules according to the
`method described in Example 9.
`
`Detailed Description of the Invention
`
`"'
`
`A.
`
`DAn
`
`Encoded Combinatorial Chemical Libraries
`encoded combinatorial chemical library is a
`composition comprising a plurality of species of
`bifunctional molecules that each define a different
`chemical structure and that each contain a unique
`~.
`Sidentifier oligonucleotide whose nucleotide sequence
`defines the corresponding chemical structure.
`25
`
`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 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
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`.. .
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`is a linker molecule operatively linked to A and C,
`and C is an identifier oligonucleotide comprising a
`sequence of nucleotides thpt 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 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 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 polymers. 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
`
`(/
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`polypeptide, can be comprised of natural D-amino
`acids, or can be comprised of non-natural amino acids
`or mixtures of natural and non-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 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. Biol. Chem., 243:3552-59 (1969) and
`adopted at 37 C.F.R. §1.822(b)(2)), abbreviations for
`amino acid residues are shown in the following Table
`
`of Correspondence:
`
`TABLE OF CORRESPONDENCE
`
`SYMBOL
`
`AMINO ACID
`
`tyrosine
`
`glycine
`phenylalanine
`methionine
`
`alanine
`serine
`isoleucine
`leucine
`threonine
`valine
`
`proline
`
`lysine
`
`L1
`
`'3-Letter
`Tyr
`Gly
`Phe
`Met
`
`Ala
`Ser
`
`Ile
`Leu
`Thr
`Val
`Pro
`
`Lys
`
`1-Letter
`Y
`
`M A S I L T V P K
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`H
`Q
`
`E
`
`W
`R
`D
`N
`C
`
`His
`Gln
`
`Glu
`
`Trp
`*Arg
`Asp
`Asn
`Cys
`
`histidine
`glutamine
`glutamic acid
`tryptophan
`arginine
`aspartic acid
`asparagine
`cysteine
`
`The phrase "amino acid residue" is broadly
`defined to include the amino acids listed in the Table
`of Correspondence and modified and unusual amino
`acids, such as those listed in 37 C.F.R. §1.822(b)(4),
`and incorporated herein by reference.
`The polymer defined by chemical moiety A cin
`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(=CHR)CO-,
`-NHC 6H4CO-,
`-NHCH2CHRCO-, -NHCHRCHzCO-, and lactam structures.
`In addition, amide bond modifications are
`contemplated including -COCH 2-, -COS-, -CONR, -COO-,
`-CSNH-,
`-CH 2NH-,
`-CH 2CH 2 -,
`-CH 2S-,
`-CH 2SO-,
`-CH 2SO2 -,
`-CH(CH 3 )S-,
`-CH=CH-,
`-NHCO-,
`-NHCONH-,
`-CONHO-,
`and
`-C (=CH 2 ) 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)a, wherein Z is a unit identifier
`nucleotide sequence within oligonucleotide C that
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`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:
`X4X3X2X1-B-Z1 Z2 Z3Z4 .
`In this example, the sequence of oligonucleotides Z1,
`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 considerations relating to
`requirements for uniqueness of oligonucleotides such
`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 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
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`reaction. However, the nucleotides A, G, T and C are
`
`CZ
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`preferred.
`For the design of the code in the identifier
`oligonucleotide, it is espential to chose a coding
`representation such that no significant part of the
`oligonucleotide sequence can occur in another
`unrelated combination by chance or otherwise 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 and permutations of an alphabet
`of chemical units, it is 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
`distinguishable by hybridization or sequencing unless
`the frame is clear.
`Other sources of misreading of a unit identifier
`oligonucleotide can arise. For example, mismatch in
`IO
`SDNA
`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 toreduce the similarity between unit
`identifier nucleotide sequences. Typical 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
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`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
`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 nucleotides in the tag (coding) region for
`effective hybridization. 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
`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 binding sites adapted to amplify
`the polymer identifier oligonucleotide. 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 VCR primer binding sites,
`P1 and P2, flanking the identifier oligonucleotide
`sequence (Zn) a provides a means to produce a PCR-
`amplified duplex DNA fragment derived from the
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`bifunctional molecule using PCR. This design is
`useful to allow the amplification of the tag sequence
`present on a particular brifunctional molecule for
`cloning and sequencing purposes in the process of
`reading 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 from the identifier
`oligonucleotide sequences. Removal of the flanking P1
`and P2 sequences is desirable so that their sequences
`do not contribute to a subsequent hybridizatioh
`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-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 specific 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-specific modifications such as incorporation
`
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`
`
`of a biotinylated nucleotide (e.g., biotinyl deoxy-
`UTP) to facilitate subsequent manipulations.
`The above described preferred embodiments in an
`identifier oligonucleotidi are summarized in a
`specific embodiment shown in Figure 1.
`In Figure 1, a PCR-amplified duplex DNA is shown
`that is derived from an identifier oligonucleotide
`described in the Examples. The (Zn) 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 located
`
`5
`
`10
`
`5' to the bracket and an Apy I restriction
`endonuclease site defined by the P2 sequence located
`
`5
`
`3' to the bracket.
`Step 1 illustrates the cleavage of the PCk-
`amplified duplex DNA by the enzymes Sty I and kpa 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 Figure 1 allows the specific ahd
`directional cloning of the restriction-digested PCR-
`amplified fragment into an appropriate vector, such as
`a sequencing vector. In addition, the Sty I was
`designed into P1 because the resulting overhang is a
`substrate for a filling-in reaction with dCTP and
`biotinyl-dUTP (BTP) using DNA polymerase KlenoW
`fragment. 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.
`
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`Page 15 of 74
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`-16-
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`Once biotinylated, the duplex fragment car be
`bound to immobilized avidin and the duplex can be
`denatured to release the coding sequence containing
`the identifier nucleotide 'sequence, thereby providing
`purified anticoding strand that is useful as a
`hybridization reagent for selection of related coding
`strands as described further herein.
`
`Linker Molecules
`c.
`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 thit
`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 bifunctional 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 as
`
`described herein. Particularly preferred are linker
`molecules 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 provide at least two features: (1)
`operative linkage to chemical moiety A, and (2)
`operative linkage to identifier oligonucleotide 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
`
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`Page 16 of 74
`
`
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`-17-
`
`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 meansofor attachment to a
`solid support. That is, the bifunctional molecule is
`
`5
`
`10
`
`removable from the solid support 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 available from a
`variety of commercial sources including Glen Research
`(Herndon, VA), Bachem Biosciences, (Philadelphia, PA),
`aand Applied Biosystems (Foster City, CA). Most
`preferred are teflon supports such as that described
`in Example 2.
`
`Libraries
`2.
`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
`Slibrary defines a family of chemical diversity whose
`25
`species each have a different chemical moiety. Thus
`the library can define a family of peptides, lipids,
`oligosaccarides 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 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
`"plurality of species" in a library can be defined by
`
`35
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`Page 17 of 74
`
`
`
`V
`
`the formula Va, i.e., V to power of a (exponent a).
`represents the alphabet.size, i.e., the number of
`different chemical units X available for use in the
`chemical moiety. "a" iq an exponent to V.and
`represents the number of chemical units of X forming
`the polymer A, i.e., the length of polymer'A.
`For example, for a bifunctional molecule where
`polymer A is a peptide having a length of 6 amino
`acids, and where the amino acids utilized can be any
`of the 20 natural amino acids, the alphabet (V) is 20
`and the polymer length (a) is 6, and the library size
`is 206 or 64 million. This exemplary library' provides
`a repertoire of chemical.diversity comprising 64
`million different hexameric. polypeptides operatively
`linked to corresponding unique identifier
`oligonucleotides.
`Because the complexity of the library will
`determine the amount of a particular species bf
`bifunctional molecule relative the other species in
`the library, there are theoretical limits to the
`maximum useful complexity in a library. Therefore it
`is useful to consider how large (complex) a library
`should be. This size limit is dictated by the level
`of sensitivity for detecting the presence of a polymer
`identifier oligonucleotide after a screening procedure
`according to this invention. Detection sensitivity is
`dictated by the threshold of binding between hn
`acceptor molecule to be assayed and a bifunctional
`molecule.
`If, for example, the binding threshold it 10-6M
`(micromolar), then there must be at least one
`nanomole of each species in a library of 1 milliliter
`(ml) volume. At this threshold, a library having a
`complexity of 104 could contain 10 micromoles of each
`species. Because of the reciprocal relationship
`
`5
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`10
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`15
`
`:tea
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`itz'
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`ao7:
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`;M .
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`~g
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`" -*'
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`Page 18 of 74
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`-19-
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`5
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`between library complexity and binding threshold, more
`complex libraries are possible where the binding
`threshold is lower.
`The relative amounts of the individual
`bifunctional molecule species within the library can
`vary from about 0.2 equivalents to about 10
`equivalents, where an equivalent represents the
`average amount of a species within the library.
`Preferably each species is present in the library in
`approximately equimolar amounts.
`In a preferred embodiment, a library contains the
`complete repertoire of chemical diversity possible
`based on the mathematical combinations for a given
`* library where there is a fixed alphabet and a
`preselected number of chemical units in all species of
`5
`the library. Thus a complete repertoire is one that
`?EJ
`r' provides a source of all the possible chemical
`diversity that can be found in a .library of this
`w.
`Winvention having a fixed alphabet and chemical length.
`I0
`It is particularly preferred that a library be
`comprised of bifunctional molecules where each species
`of bifunctional molecule contains the same nucleotide
`sequence for either the P1 or P2 PCR primer binding
`sites. A library with this design is particularly
`preferred because, when practicing the methods of this
`invention, a single PCR primer pair can be used to
`amplify any species of identifier oligonucleotide
`(coding sequence) present in the library.
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`
`B. Methods for Producing a Library
`The present method for producing a plurality of
`bifunctional molecules to form a library of this
`invention solves a variety of problems regarding
`efficient synthesis of large numbers of different
`species.
`
`t/
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`Page 19 of 74
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`
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`-20-
`
`In the present synthesis methods, the sequential
`steps of first adding a chemical unit X followed by
`the addition of an oligonuqleotide sequence to the
`linker molecule requires an alternating parallel
`synthesis procedure to add chemical unit X and then
`
`add a unit identifier nucleotide sequence Z that
`
`defines (codes for) that corresponding chemical unit.
`The library is built up by the repetition of this
`alternating parallel process after pooling and
`division of the reaction products as described herein.
`The only constraint for making an encoded library
`is that there must be compatible chemistries between
`the two alternating syntheses procedures for adding a
`.chemical unit as compared to that for adding a
`nucleotide or oligonucleotide sequence.
`The problem. of synthesis compatibility is solved
`by the correct choice of compatible protecting groups
`as the alternating polymers are synthesized, and by
`the correct choice of methods for deprotection of one
`growing polymer selectively while the other growing
`polymer remains blocked.
`The synthesis of a library having a plurality of
`bifunctional molecules comprises the following steps:
`(1) A linker molecule is provided that has
`suitable means for operatively linkin
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