Description
`
`mmm
`
`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.\
`
`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 fermentation broths or
`plant extracts, or libraries of synthesized molecules
`using assays which can range in complexity from simple
`binding reactions to elaborate physiological
`
`The screens often only provide leads
`preparations.
`which then require further improvement either by
`
`The process
`empirical methods or by chemical design.
`‘it time—consuming and costly but it is unlikely to be
`
`10
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`15
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`20
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`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
`
`30
`
`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
`
`35
`
`compounds for lead discovery.
`
`The methods were
`
`Page1 of67
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`ILMN EXHIBIT 1006
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`
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`Page 1 of 67
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`ILMN EXHIBIT 1006
`
`
`
`mmm
`
`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.\
`
`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 fermentation broths or
`plant extracts, or libraries of synthesized molecules
`using assays which can range in complexity from simple
`binding reactions to elaborate physiological
`
`The screens often only provide leads
`preparations.
`which then require further improvement either by
`
`The process
`empirical methods or by chemical design.
`‘it time—consuming and costly but it is unlikely to be
`
`10
`
`15
`
`20
`
`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
`
`30
`
`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
`
`35
`
`compounds for lead discovery.
`
`The methods were
`
`Page1 of67
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`ILMN EXHIBIT 1006
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`Page 1 of 67
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`ILMN EXHIBIT 1006
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`
`
`example,
`
`l process of stepwise
`s a variety
`
`epitopes recognized by
`the standard seria
`ic peptides now encompasse
`"search of synthet
`I
`hich large arrays.
`isticated methods in w
`ized in paralle
`lled with fluorescent or
`e of any effective
`
`l and screened
`
`of highly soph
`of peptides are synthes
`tor molecules labe
`with accep
`other reporter groups.
`The sequenc
`dress in the array.
`peptide can be decoded from its ad
`See for example Geysen et al., Proc.Natl.Acad.Sci.USA,
`8l:3998~4002 (1984); Maeji et al.,
`l46:83w9O (1992); and Fodor et al.,
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`J.Immunol.Met.,
`
`Science, 251: 767-
`
`775 (1991).
`al., Nature, 354:82—
`In another approach, Lam et.
`84 (1991) describes combinatorial libraries of
`peptides that are synthesized on resin beads suc
`each resin bead contains about 20 pmoles of the same
`The beads are screened with labelled
`peptide.
`acceptor molecules and those with bound acceptor are
`r by visual inspection, physically removed,
`searched fo
`and the peptide identified by dir
`analysis.
`In principle,
`this method could be used
`ut it requires sensitive
`with other chemical entities b
`ination.
`methods for sequence determ
`the problem of
`A different method of solving
`binatorial peptide library is
`Nature, 354:84—86 (1991).
`
`h'that
`
`ect sequence
`
`identification in a com
`used by-Houghten et al.,
`For hexapeptides of the 20 natura
`separate libraries are synthesized,
`two amino acids fixed and the rem
`e combinations.
`itions occupied by all possibl
`based on competition for binding or other
`nd the library with an
`is then used to fi
`
`aining four
`
`An
`
`assay,
`
`activity,
`
`active peptide.
`synthesized and assayed to determ
`
`Then twenty new
`
`libraries are
`
`ine the effective
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`Page2of67
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`x_
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`H,
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`Page 2 of 67
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`
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`_..3_.
`
`amino acid in the third position, and the process is
`reiterated in this fashion until the active
`
`the peptide is
`method used in searching a dictionary;
`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
`followed by their
`
`Each of the sequences
`is cloned in one phage and the relevant peptide can be
`selected by finding those that bind to the particular
`The phages recovered in this way can be
`target;
`The sequence of
`amplified and the selection repeated.
`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
`
`(1990).
`method has been described where
`Another "geneticfi
`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).
`allows serial enrichment and the structure of the
`active molecules is then decoded by DNA sequencing on
`clones generated from the PCR products.
`The
`
`PCR
`
`repertoire
`-pyrimidine and purine bases or those modifications
`that preserve specific Watson—Crick pairing and can be
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`copied by polymerases.
`
`lflf
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`Pmesmer
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`Page 3 of 67
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`-4...
`
`ges of the genetic methods reside
`"The main advanta
`lification of DNA
`r cloning and amp
`erial~selection
`hment by s
`
`in the capacity fo
`h allows enric
`sequences,_whic
`acile method for decod
`and provides a-f
`structure of active molecules.
`repertoires are restricted to nucl
`ed of natural amino acid
`
`10
`
`unimethods can provide limitless repertoires but they
`lack the capacity for serial enrichment and there are
`
`selected
`
`
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`the virtues o
`
`f the Invention
`a way of combining
`Brief Summary o
`The present invention provides
`f both of the chemical and genetic
`methods summarized above through the construction of
`braries,
`in which
`encoded combinatorial chemical li
`led by an appended
`each chemical sequence is label
`"genetic" tag,
`itself constructe
`‘synthesis,
`to provide a "retrogenetic" way of
`specifying each c
`In outline,
`
`d by chemical
`
`hemical structure.
`two alternating parallel
`s are performed so that
`
`in each case,
`ical units to
`
`the struct
`
`genetic tag is chem
`structure being synthesized:
`addition of one of the particular chem
`ure is followed by the addition of an
`which is defined to "code"
`ucleotide sequence,
`oligcn
`to function as an
`ie.,
`for that chemical unit,
`re of the chemical unit.
`identifier for the structu
`The library is built up b
`tition of this
`y the repe
`process after pooling and
`Active molecules are se
`
`division.
`lected from the librar
`
`y so
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`Page 4 of 67
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`Page 4 of 67
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`
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`f5¥
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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
`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 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
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`of chemical diversity.
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`Page5of67
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`Page 5 of 67
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`...6..
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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
`library of bifunctional molecules
`The method comprises the
`
`present in the
`according to this invention.
`
`steps of:
`
`admixing in solution the library of
`a)
`y active
`bifunctional molecules with the biologicall
`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)
`
`determinin
`
`g the nucleotide sequence of
`gonucleotide in the isolated
`the polymer identifier oli
`complex and thereby identifying the chemical structure
`that participated in the preselected binding
`
`interaction.
`The invention also contemplates a method for
`
`comprising the steps of:
`a)
`providing a linker molecule B having
`and C‘ according to the formula K‘—B-C‘
`termini A‘
`a chemical precursor
`that is adapted for reaction with
`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 pre
`cursor unit identifier oligonucleotide Z‘
`of said linker,
`to form a composition
`e structure
`containing bifunctional molecules having th
`
`to
`
`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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`1r
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`1II|
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`-._'7_
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`.d)
`
`combining the aliquots produced in step
`
`(c)
`
`to form an admixture of bifunctional molecules,
`
`thereby forming said library.
`
`Brief 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
`
`10
`
`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 the process of producing a
`
`library of bifunctional molecules according to the
`
`15
`
`method described in Example 9.
`
`Detailed Description of the Invention
`
`A.
`
`Encoded Combinatorial Chemical Libraries
`
`20
`
`An 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
`
`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 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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`K >
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`Page 7 of 67
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`.-8-
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`is a linker molecule operatively linked to A and C,
`ntifier oligonucleotide comprising a
`and C is an ide
`he structure
`sequence of nucleotides that identifies t
`of chemical moiety A.
`
`_
`Chemical Polymers '
`a.
`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
`
`1
`
`X is
`
`Polymer A
`
`having
`
`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 O to 10,
`located most proximal to the linker (B).
`Although the length of the polyme
`practical library size limitations arise
`defined by a,
`e as discussed further
`if there is a large alphabet siz
`herein. Typically,
`r from 4 to 50.
`a is an intege
`polymer A) can be any of a
`A chemical moiety (
`variety of polymeric structures, depending on the
`choice of classes of chemical diversity to be
`represented in a library of this invention.
`coupled
`can be any mon
`omeric chemical unit that can be
`For_example, polymer
`and extended in polymeric form.
`A can be a polypeptide, oligosaccharide, glycolipid,
`lipid, proteoglycan, glycopeptide, sulfonamide,
`conjugated peptide (i.e.,
`nucleoprotein,
`polymer containing enzyme
`nsition state analogues, and
`Exemplary is’the,
`the like biochemical polymers.
`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 nonwnatural
`
`prosthetic groups),
`substrates,
`including tra
`
`Page8of67
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`U]
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`,_9_,
`
`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 Dwamino
`
`acids.
`The amino acid residues described herein are
`preferred to be in the "L" isomeric form.
`NH; refers
`to the free amino group present at the amino terminus
`of a polypeptide.
`'CO0H 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. §l.822(b)(2)), abbreviations for
`amino acid residues are shown in the following Table
`
`of Correspondence:
`
`______H_jflflE&EL#__#__H
`
`AMINQ ACID
`
`TABLE OF CORRESPONDENCE
`
`l—Letter
`Y
`G
`F
`M
`A
`S
`I
`L
`T
`V
`
`P
`
`K
`
`3—Letter
`Tyr
`'
`Gly
`Phe
`Met
`Ala
`Ser
`Ile
`Leu
`Thr
`Val
`
`Pro
`
`LYS
`
`tyrosine
`glycine
`phenylalanine
`methionine
`alanine
`serine
`isoleucine
`leucine
`threonine
`valine
`
`proline
`
`lysine
`
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`Page 9 of 67
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`...:]_O_.
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`'
`
`H
`
`Q
`E
`
`w
`
`R
`D
`
`N
`
`His
`
`Gln
`Glu
`
`Trp
`
`Arg
`Asp
`
`Asn
`
`_ histidine
`
`glutamine
`glutamic acid
`
`tryptophan
`
`arginine
`aspartic acid
`
`asparagine
`
`cysteine
`.
`Cys
`C
`
`
`“io
`
`‘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. §l.822(b)(45,
`
`15
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`35
`
`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: wNHN(R)CO~,
`
`—NHB(R)CO~,
`
`-NHC(RR')CO-,
`
`-NHC(=CHR)CO—,
`
`“NHC§fiCO~,
`
`—NHCH3CHRCO—, —NHCHRCH2CO~, and lactam structures.
`
`In addition, amide bond modifications are
`
`contemplated including —COCH2-, —COS—, —CONR,
`
`-COO—,
`
`-cH2so2-,
`-CSNH-, —cH2NH—-, —CH2CI-I2-~, —CH2S-, —CI-I280-,
`-—CH(CH3)S-, —CH=CH-,
`-NHCO-,
`-NI-ICON},-I--,
`-CONHO-, and
`
`-C (=CI-I2) 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 (zpa, wherein Z is a unit identifier
`
`nucleotide sequence within oligonucleotide C that
`
`Page10of67
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`Page 10 of 67
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`_11-
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`n has
`identifies the chemical unit Xiat position n.
`the value of l+i where i is an integer from O 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:
`
`X,,x3x2x,—B—z,z2z3z,,'.
`y
`In this example,
`the sequence of oligonucleotides Z“
`Z2,
`Z3 and Z4 identifies the structure of chemical
`units X1, X3, 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
`as hybridization and polymerase chain reaction
`A typical length can be from about 2 to
`
`fidelity.
`
`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
`Other
`
`identifier oligonucleotide's sequence.
`
`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
`
`reaction. However,
`
`the nucleotides A, G, T and C are
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`_l2.-.
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`preferred.
`For the design of the code in the identifier
`oligonucleotide, 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 otherwise during
`the manipulations of a bifunctional molecule in the
`
`library.
`I
`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
`
`For example, mismatch in‘
`oligonucleotide can arise.
`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
`
`.5
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`25
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`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
`
`30
`
`sequence as to reduce 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
`
`35
`
`developed as a theoretical alternative to the genetic
`
`Page12of67
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`Page 12 of 67
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`_:L3...
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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—(Zga~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 PCR primer binding sites,
`P1 and P2,
`flanking the identifier oligonucleotide
`
`sequence (zna provides a means to produce a PCR-
`amplified duplex DNA fragment derived from the
`
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`-14..
`
`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 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 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—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.
`the restriction endonuclease sites
`Preferably,
`should be positioned proximal to (293 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
`terminiuspecific modifications such as incorporation
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`Page 14 of 67
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`
`
`—15~
`
`of a biotinylated nucleotide (e.g., biotinyl deoxy—
`UTP) to-facilitate subsequent manipulations.
`The above described preferred embodiments in an
`identifier oligonucleotide 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 Pl sequence located
`5'
`to the bracket and an Apy I restriction
`endonuclease site defined by the P2 sequence located
`3'
`to the bracket.
`‘
`V
`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 Figure 1 allows the specific and
`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 Pl because the resulting overhang is a
`substrate for a fillingflin 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
`
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`be biotinylated.
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`‘-16-
`
`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 nucleotide sequence,
`
`thereby providing
`
`5
`
`purified anticoding strand that is useful as a
`
`hybridization reagent for selection of related coding
`
`strands as described further herein.
`
`10
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`15
`
`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 bifunctional molecule in the solid
`
`phase.
`
`In addition, attachment to a solid support
`
`provides certain features in practicing the screening
`
`20
`
`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.
`
`25
`
`- 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
`
`linkages is diverse, any of a
`the nature of chemical
`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 of67
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`Page 16 of 67
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`
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`—l7_.
`
`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 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),
`and Applied Biosystems (Foster City, CA). 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,
`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
`
`10
`
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`
`
`
`Page 17 of 67
`
`
`
`o
`the formula V“, i.e., V to power of a (exponent a).
`
`V
`
`represents the alphabet size, i.e.,
`
`the number of
`
`different chemical units X available for use in the
`
`chemical moiety.
`
`"a" is 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
`
`V
`
`oligonucleotides.
`
`Because the complexity of the library will
`
`determine the amount of a particular species of
`
`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 an
`
`acceptor molecule to be assayed and a bifunctional
`
`molecule.
`
`If,
`
`for example,
`
`the binding threshold is lO*M
`
`(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 lo‘ could contain 10 micromoles of each
`
`species. Because of the reciprocal relationship
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`Page 18 of 67
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`
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`_19—.
`
`between library complexity and-binding threshold, more
`complex libraries are possible where the binding
`
`threshold is lower.
`
`The relative amounts of the individual
`
`5'
`
`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
`
`10
`
`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
`
`15
`
`preselected number of chemical units in all species of
`the library.
`Thus a complete repertoire is one that
`
`provides a source of all the possible chemical
`
`diversity that can be found in a library of this"
`
`invention having a fixed alphabet and chemical
`
`length.
`
`20
`
`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
`V preferred because, when practicing the methods of this
`
`25
`
`
`
`invention, a single PCR primer pair can be used to
`
`amplify any species of identifier oligonucleotide
`
`(coding sequence) present in the library.
`
`30
`
`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
`
`35
`
`species.
`
`Page 19cfi67 ,___m_
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`mm__m_
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`Page 19 of 67
`
`
`
`-20..
`
`the sequential
`In the present synthesis methods,
`steps of first adding a chemical unit X followed by
`the addition of an oligonucleotide 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
`
`U1
`
`10
`
`15
`
`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
`
`20
`
`growing polymer selectively while the other growing
`
`25
`
`30
`
`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 linking the first
`chemical_unit X1 and for operatively linking the first
`
`nucleotide sequence defining a unit identifier
`
`nucleotide Z1 whose sequence codes for (defines) the
`
`structure of chemical unit X1. Preferably the linker
`
`has a means for attachment to a solid support, and as
`
`such allows for the synthesis to p
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