Ulllted States Patent [19]
`Dower et al.
`
`US006143497A
`[11] Patent Number:
`[45] Date of Patent:
`
`6,143,497
`*Nov. 7, 2000
`
`[54] METHOD OF SYNTHESIZING DIVERSE
`COLLECTIONS ()F ()LIGOMERS
`
`5,264,563 11/1993 Huse .................................... .. 536/25.3
`5,708,153
`1/1998 Dower et al. ........................ .. 536/221
`
`[75] Inventors: William J. Dower, Menlo Park;
`Ronald W. Barrett, Sunnyvale; Mark
`A. Gallop, E. Palo Alto, all of Calif.
`
`[73] Assignee: A?'ymax Technologies N.V., Greenford,
`
`FOREIGN PATENT DOCUMENTS
`0 392 546 A2 12/1990 European Pat. Off. ........ .. C12Q 1/68
`WO 90/00626
`1/1990 WIPO ............................ .. C12Q 1/68
`WO 90/14441 11/1990 WIPO ............................ .. C12Q 1/68
`W0 90/15070 12/1990 WIPO
`C07K 1/04
`
`United Kingdom
`
`WO 92/00091
`
`1/1992 WIPO . . . . .
`
`. . . .. A61K 37/02
`
`WO 92/03461
`
`3/1992 WIPO . . . . .
`
`. . . .. CO7H 17/00
`
`4
`
`[
`
`~
`
`.
`
`] Nome‘
`
`~
`
`~
`
`~
`
`~
`
`-
`
`glgliingitem 15 Subject to a termmal dls'
`'
`
`WO 92/06176
`
`4/1992 WIPO ............................ .. C12N 1/24
`
`WO 92/10092 6/1992 WIPO ............................ .. AO1N 1/02
`WO 93/20242 10/1993 WIPO
`C12Q 1/70
`WO 94/02515
`2/1994 WIPO
`. C07K 17/06
`WO 94/08051
`4/1994 WIPO ............................ .. C12Q 1/68
`
`OTHER PUBLICATIONS
`
`Amato, 1992, Science, 257:330—331 Speeding up a chemi
`cal game of chance.
`Hodgson, 1992, Bio/Technology 10Z973—974 Receptor
`Screening and the Search for new pharmaceuticalg
`Mathur et al., 1980,P0lymers asAia's in Organic Chemistry,
`Academic Press, New York, Ch- 9—12r138—197
`Needels et al., 1993, Proc. Natl. Acad. Sci, USA,
`9011070910704 Generation and screening of an Oligo
`nucleotide—encoded sythetic peptide library.
`Advances in Analytical Chemistry and Instrumentation,
`Charles N. Reilley, Ed., John Wiley & Sons, Inc., 1964, pp.
`56—59.
`
`[21] Appl. No.: 09/036,599
`
`[22] Filed:
`
`Mar. 6, 1998
`
`Related US Application Data
`
`[51]
`
`[63] Continuation of application No. 08/484,085, Jun. 7, 1995,
`abandoned, Which is a continuation of application No.
`07/762522’ Sep' 18’ 1991’ abandoned‘
`Int. Cl.7 ............................. .. C12Q 1/68; C12Q 1/70;
`C12P 19/34
`[52] US. Cl. ................................ .. 435/6;435/5;435/91.1;
`435/91_2; 536/243; 536/265
`[58] Field of Search ................................. .. 435/6, 5, 91.1,
`435/912; 536/243, 2&6
`
`[56]
`
`References Clted
`
`(List continued on neXt page.)
`
`US. PATENT DOCUMENTS
`
`4,182,654
`
`1/1980 Royer .................................... ..
`
`_
`_
`_
`Primary Examiner—Kenneth R. Horlick
`Assistant Examiner_J_ Tung
`
`473157074
`4,587,044
`
`2/1982 ROYer - - - - - - - -
`5/1986 Miller et al. ..
`
`- - - - ~~ 435/70
`530/211
`
`Attorney, Agent, or Firm—Lauren L. Stevens; Kevin R.
`Kaster
`
`4,631,211 12/1986 Houghten . . . . . .
`
`. . . . .. 428/35
`
`422/131
`6/1987 Niina et al.
`4,671,941
`4,701,304 10/1987 Horn et al. .............................. .. 422/62
`4,713,326 12/1987 Dattagupta et al. ...................... .. 435/6
`4J755J558
`7/1988 Kalbag ~~~~~~~~~~~~ ~~
`525/54-1
`47947150 12/1988 Steel ~~~~~~~~ ~~
`- 525/5411
`gzttsegrlllpta
`""
`8/1989 Furlg et al. ................................ .. 435/6
`4,855,225
`4,965,188 10/1990 Mullis et al.
`435/6
`5,143,854
`9/1992 Pirrung et al. ........................ .. 436/516
`
`[57]
`
`ABSTRACT
`
`A general stochastic method for synthesizing random oli
`gomers on particles is disclosed. A further aspect of the
`invention relates to the use of identi?cation tags on the
`particles to facilitate identi?cation of the sequence of the
`monomers 1“ the Ohgomer'
`
`21 Claims, 6 Drawing Sheets
`
`we
`/[
`]_1
`0.]
`LL]
`1
`l
`l
`%
`i8
`i8
`\‘Piof/
`
`n EACH
`
`of 9% 9% 9 9
`
`H
`
`t
`
`[ml
`
`9555mm
`
`
`
`Page 1 of 24
`
`ILMN EXHIBIT 1008
`
`

`
`6,143,497
`Page 2
`
`OTHER PUBLICATIONS
`
`Arnold et al., Mar. 15, 1991, Optics Letters 16(6):420—422
`Room—temperature microparticle—based persistent spectral
`hole burning memory.
`Baldwin et al., 1990, Tetrahedron 46(19):6879—6884 NeW
`photolabile phosphate protecting groups.
`Barr
`et
`al.,
`1986, BioTechniques 4(5):428—432,
`7—DeaZa—2‘—deoXyguanosine—5‘—triphosphate: Enchanced
`resolution in M13 dideoXy sequencing.
`Bashkin et al., 1991,]. Org. Chem. 56:3168—3176 Synthesis
`and characterization of oligonucleotide peptides.
`Brenner et al., 1992, Proc. Natl. Acad. Sci. USA
`89:5381—5383 Encoded combinatorial chemistry.
`CWirla et al., 1990, Proc. Natl. Acad. Sci. USA
`87:6378—6382 Peptides on phage: Avast library of peptides
`for indentifying ligands.
`Eritja et al., 1991, Tetrahedron 47(24):4113—4120, Synthesis
`of de?ned peptide—oligonucleotide hybrids containing a
`nuclear transport signal sequence.
`Fodor et al., 1991, Science 251:767—773 Light—directed,
`spatially addressable parallel chemical synthesis.
`Frank and Doring, 1988, Tetrahedron 44(19):6031—6040
`Simultaneous multiple peptide synthesis under continuous
`?oW conditions on cellulose paper discs as segmental solid
`supports.
`Frank et al., 1983, Nuc. Acids. Res. 11(13):4365—4377 A
`neW general approach for the simultaneous chemical syn
`thesis of large numbers of oligonucleotides: Segmental solid
`supports.
`Frank et al., 1990, Peptides (Giralt & Andreu, eds., ESCOM
`Science Pub.), pp. 151—152 Facile and rapid ‘spot—synthe
`sis’ of large numbers of peptides on membrane sheets.
`Furka et al., Jul. 10—15, 1988, 14th Intl. Congress of
`Biochemistry, Prague, Czechoslovakia, Abstract No. FR:013
`Proteins and nucleic acids in threee dimensions: Cornucopia
`of peptides by synthesis.
`Furka et al., Aug. 15—19, 1988,Xth Intl. Symp. on Medicinal
`Chem., Budapest, Hungary, Abstract No. P—168, p. 268
`More peptides by less labour.
`Furka et al., 1991, Int. ]. Peptide Protein Res., 37:487—493
`General method for rapid synthesis of multicomponent
`peptide mixtures.
`Geysen et al., 1987,]. Immunol. Meth., 102:259—274 Strat
`egies for epitope analysis using peptide synthesis.
`
`Geysen et al., 1984, Proc. Natl. Acad. Sci. USA,
`81:3998—4002 Use of peptide synthesis to probe viral anti
`gens for epitopes to a resoution of a single amino acid.
`Haralambidis et al., 1990, Nuc. Acids Res. 18(3):493—505
`The synthesis of polyamide—oligonucleotide conjugate mol
`ecules.
`HayakaWa et al., 1990, ]. Am. Chem. Soc. 112:1691—1696
`The allylic protection method in solid—phase oligonecleotide
`synthesis: An ef?cient preparation of solid—anchored DNA
`Oligomers.
`Houghten et al., 1991, Nature 354:84—86 Generation and use
`of synthetic peptide combinatorial libraries for basic
`research and drug discovery.
`Houghten, 1985, Proc. Natl. Acad. Sci. USA 82:5131—5135
`General method for the rapid solid—phase synthesis of large
`numbers of peptides: Speci?city of antigen—antibody inter
`action at the level of individual amino acids.
`Juby et al., 1991, Tetrahedron Letters 32(7):879—882 Facile
`preparation of 3‘ oligonucleotide—peptide conjugates.
`Kaiser et al., 1989, Science 243:187—192 Peptide and pro
`tein synthesis by segment synthesis—condensation.
`Kerr et al., 1993,]. Am Chem Soc., 115:2529—2531 Encoded
`combinatorial peptide libraries containing non—natural
`amino acids.
`Lam et al., 1991, 12th Amer Pep. Symp., Abstract LW3
`Rapid selection and structure determination of acceptor
`binding ligands from a large synthetic peptide library.
`Lam et al., 1991, Nature 354:82—84 A neW type of synthetic
`peptide library for indentifying ligand—binding activity.
`Nikolaiev et al., 1993, Peptide Research, 6(3):161—170
`Peptide—encoding for structure determination of nonse
`quenceable polymers Within libraries synthesiZed and tested
`on solid phase supports.
`Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA,
`90:10922—10926 Complex synthetic chemical libraries
`indeXed With molecular tags.
`Tjoeng et al., 1990, Int. ]. Pept. Protein Res. 35:141—146
`Multiple peptide synthesis using a single support (MPS3).
`Van der Zee et al., 1989,Eur ]. Immunol. 19:43—48 Ef?cient
`mapping and characteriZation of a T cell epitope by the
`simultaneous synthesis of multiple peptides.
`
`
`
`Page 2 of 24
`
`

`
`U.S. Patent
`
`Nov. 7,2000
`
`Sheet 1 of6
`
`6,143,497
`
`Page 3 of 24
`
`
`
`Page 3 of 24
`
`

`
`U.S. Patent
`
`Nov. 7,2000
`
`Sheet 2 0f6
`
`6,143,497
`
`COUPLE MONOMER
`
`COUPLE STEP-SPECIFIC TAC
`
`COUPLE MONOMER
`
`COUPLE STEP-SPECIFIC TAC
`
`[ME
`0 EACH
`[Mir
`gqnfyg)
`9
`
`COUPLE MONOMER
`
`COUPLE STEP-SPECIHC TAC
`
`
`
`Page 4 of 24
`
`

`
`U.S. Patent
`
`Nov. 7,2000
`
`Sheet 3 0f6
`
`6,143,497
`
`(ii) PIPERIDINE
`
`FIG: 3A
`
`
`
`Page 5 of 24
`
`

`
`U.S. Patent
`
`Nov. 7,2000
`
`Sheet 4 of6
`
`6,143,497
`
` NH ]
`2
`
`n
`
`+
`
`+
`
`-H
`Fmoc NW/COZH
`R1
`
`99.9
`
`H
`FmocN\(002l1—-GD
`5-5
`N
`0.!
`
`1
`
`HOBt,BOP
`
`0
`
`H
`
`fi)'\(NFmoc ]
`
`FmocNI/LL”
`s-sf?
`
`Y
`
`I000
`
`0
`H
`N ,U\l,NFmoc
`H
`R‘
`
`x
`
`Y
`
`DTT
`
`
`
`Y
`
`0
`
`N H
`
`SH
`
`H
`Fmodl
`
`FIG 3 C
`
`Page 6 of 24
`
`
`
`Page 6 of 24
`
`

`
`U.S. Patent
`
`Nov. 7,2000
`
`Sheet 5 of6
`
`6,143,497
`
`//@"""AA| -Fmoc
`
`P
`
`[once]!
`
`FIG 30
`
`Page 7 of 24
`
`
`
`Page 7 of 24
`
`

`
`I
`
`Sheet 6 of 6
`
`6,143,497
`
`PIPERIDINE
`(i)
`
`
`(99.9)
`(ii) Fmoc~AA2-OH
`Fmoc-Cys(Npg5)-OH (M)
`H031 , BOP
`
`(iii) DTT
`
`“"’
`
`0:0: N~v~[0LIGO:|:
`
`0
`
`[0LlG0]: AA, -AA2-Fmoc
`[ouctflf
`
`F/GI 3E
`
`P
`
`[ouco] n
`
`\\?-AA.---AA" -Fmoc
`
`
`
`m THIOPHENOL, NR3,
`(an m, cH2Cl2
`um ETHYLENEDIAMINE, ETHANOL, A
`
`DIOXANE
`
`P I
`
`[ouco]
`
`[0LlG0]n
`
`[ouco],
`
`/'76.‘ 3F
`
`3' 5'
`._,,..__..
`I
`2
`3
`456
`7
`
`FIG 4.
`
`Page 8 of 24
`
`
`
`Page 8 of 24
`
`

`
`1
`METHOD OF SYNTHESIZING DIVERSE
`COLLECTIONS OF OLIGOMERS
`
`6,143,497
`
`2
`Prior methods of preparing large numbers of different
`oligomers have been painstakingly sloW When used at a
`scale suf?cient to permit effective rational or random screen
`ing. For example, the “Merri?eld” method (J. Am. Chem.
`Soc. (1963) 85:2149—2154, Which is incorporated herein by
`reference) has been used to synthesiZe peptides on a solid
`support. In the Merri?eld method, an amino acid is
`covalently bonded to a support made of an insoluble poly
`mer. Another amino acid With an alpha protected group is
`reacted With the covalently bonded amino acid to form a
`dipeptide. After Washing, the protective group is removed
`and a third amino acid With an alpha protective group is
`added to the dipeptide. This process is continued until a
`peptide of a desired length and sequence is obtained. Using
`the Merri?eld method, it is not economically practical to
`synthesiZe more than a handful of peptide sequences in a
`day.
`To synthesiZe larger numbers of oligomer sequences, it
`has also been proposed to use a series of reaction vessels for
`oligomer synthesis. For example, a tubular reactor system
`may be used to synthesiZe a linear oligomer on a solid phase
`support by automated sequential addition of reagents. This
`method still does not enable the synthesis of a suf?ciently
`large number of oligomer sequences for effective economi
`cal screening.
`Methods of preparing a plurality of oligomer sequences
`are also knoWn in Which a foraminous container encloses a
`knoWn quantity of reactive solid supports, the solid supports
`being larger in siZe than openings of the container. The
`containers may be selectively reacted With desired materials
`to synthesiZe desired sequences of product molecules. As
`With other methods knoWn in the art, this method cannot
`practically be used to synthesiZe a suf?cient variety of
`polypeptides for effective screening.
`Other techniques have also been described. These meth
`ods include the synthesis of peptides on 96 plastic pins
`Which ?t the format of standard microtiter plates.
`Unfortunately, While these techniques have been someWhat
`useful, substantial problems remain. For example, these
`methods continue to be limited in the diversity of sequences
`Which can be economically synthesiZed and screened.
`From the above, it is seen that an improved method and
`apparatus for synthesiZing a diverse collection of chemical
`sequences is desired.
`
`SUMMARY OF THE INVENTION
`
`The present invention provides a general stochastic
`method for synthesiZing random oligomers on solid
`supports, or particles. The oligomers are composed of a
`sequence of monomers, the monomers being any member of
`the set of molecules Which can be joined together to form an
`oligomer or polymer, i.e. amino acids, nucleic acids,
`carbohydrates, lipids, polyesters, and the like. The method
`involves producing a large library of solid supports, each
`support having attached a single oligomer sequence, the
`oligomers being synthesiZed in a random combinatorial
`(“stochastic”) fashion. The library is then screened to isolate
`individual solid supports carrying oligomers that bind to a
`receptor. Each oligomer sequence in the library is unique, in
`a preferred embodiment. In another preferred embodiment,
`the solid supports are nonporous beads. The solid supports
`may be composed of a single particle, or tWo or more linked
`particles.
`A further embodiment of the invention is the use of an
`identi?er tag to identify the sequence of monomers in the
`oligomer. The identi?er tag, Which may be attached to the
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`This application is a continuation of Ser. No. 08/484,085,
`?led Jun. 7, 1995, noW abandoned, Which is a continuation
`of Ser. No. 07/762,522, ?led Sep. 18, 1991, noW abandoned.
`FIELD OF THE INVENTION
`The present invention relates generally to a general sto
`chastic method for synthesizing random oligomers on par
`ticles. A further aspect of the invention relates to the use of
`identi?cation tags on the particles to facilitate identi?cation
`of the oligomer sequence.
`BACKGROUND OF THE INVENTION
`The relationship betWeen structure and activity of mol
`ecules is a fundamental issue in the study of biological
`systems. Structure-activity relationships are important in
`understanding, for example, the function of enzymes, the
`Ways in Which cells communicate With each other, as Well as
`cellular control and feedback systems. Certain macromol
`ecules are knoWn to interact and bind to other molecules
`having a very speci?c three-dimensional spatial and elec
`tronic distribution. Any large molecule having such speci
`?city can be considered a receptor, Whether it is an enZyme
`catalyZing hydrolysis of a metabolic intermediate, a cell
`surface protein mediating membrane transport of ions, a
`glycoprotein serving to identify a particular cell to its
`neighbors, an IgG-class antibody circulating in the plasma,
`an oligonucleotide sequence of DNA in the nucleus, or the
`like. The various molecules Which receptors selectively bind
`are knoWn as ligands.
`Many assays are available for measuring the binding
`af?nity of knoWn receptors and ligands, but the information
`Which can be gained from such experiments is often limited
`by the number and type of ligands Which are available.
`Novel ligands are sometimes discovered by chance or by
`application of neW techniques for the elucidation of molecu
`lar structure, including x-ray crystallographic analysis and
`recombinant genetic techniques for proteins.
`Small peptides are an exemplary system for exploring the
`40
`relationship betWeen structure and function in biology. A
`peptide is a sequence of amino acids. When the tWenty
`naturally occurring amino acids are condensed into poly
`meric molecules they form a Wide variety of three
`dimensional con?gurations, each resulting from a particular
`amino acid sequence and solvent condition. The number of
`possible pentapeptides of the 20 naturally occurring amino
`acids, for example, is 205 or 3.2 million different peptides.
`The likelihood that molecules of this siZe might be useful in
`receptor-binding studies is supported by epitope analysis
`studies shoWing that some antibodies recogniZe sequences
`as short as a feW amino acids With high speci?city.
`Furthermore, the average molecular Weight of amino acids
`puts small peptides in the siZe range of many currently
`useful pharmaceutical products. Of course, larger peptides
`may be necessary for many purposes; and polypeptides
`having changes in only a small number of residues may also
`be useful for such purposes the analysis of structure-activity
`relationships.
`Pharmaceutical drug discovery is one type of research
`Which relies on such a study of structure-activity relation
`ships. In most cases contemporary pharmaceutical research
`can be described as the process of discovering novel ligands
`With desirable patterns of speci?city for biologically impor
`tant receptors. Another example is research to discover neW
`compounds for use in agriculture, such as pesticides and
`herbicides.
`
`45
`
`50
`
`55
`
`60
`
`65
`
`
`
`Page 9 of 24
`
`

`
`3
`same particle as the oligomer or to a second particle attached
`to the oligomer-carrying particle, may be any recognizable
`feature that in some Way carries the required information,
`and that is decipherable at the level of one or a feW solid
`supports. The solid supports may-be joined to the oligomers
`and the identi?er tag by means of a linker molecule.
`In a preferred embodiment, the identi?er tag Will be an
`oligonucleotide, preferably composed of pyrimidines. The
`oligonucleotide identi?er tag may contain a 5‘ and a 3‘
`ampli?cation site, to alloW ampli?cation of the tag by, for
`eXample, polymerase chain reaction. A DNA sequencing
`primer site, Which may be speci?c for each step of the
`oligomer synthesis, may also be included in the oligonucle
`otide tag. The tag may be designed to include, in the
`oligonucleotide sequence, information alloWing identi?ca
`tion of the monomer associated With the addition of the
`particular tag. The oligonucleotide Will be about 100 nucle
`otides in length, in a preferred embodiment.
`
`10
`
`15
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`20
`
`FIG. 1 is a schematic representation of combinatorial
`oligomer synthesis on particles.
`FIG. 2 is a schematic representation of concurrent com
`binatorial oligomer synthesis and particle tagging.
`FIG. 3 is a description of one method of bead
`functionaliZation, the compatible chemistries for peptide
`synthesis and round by round attachment of oligonucleotide
`identi?er tags, including synthesis of amino-functionaliZed
`beads, shoWn in FIG. 3A the structure of protected 5‘
`maleimidyl oligonucleotides, shoWn in FIG. 3B amino acid
`coupling and introduction of a thiol “handle,” shoWn in FIG.
`3C step-speci?c oligonucleotide attachment to a bead,
`shoWn in FIG. 3D subsequent amino acid coupling(s) and
`oligonucleotide attachment(s), shoWn in FIG. 3E and pep
`tide and oligonucleotide deprotection, shoWn in FIG. 3F.
`FIG. 4 is a schematic representation of one eXample of an
`oligonucleotide tag.
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`The present invention provides novel methods and instru
`ments for producing large synthetic oligomer libraries. In a
`preferred embodiment of the present invention, each mem
`ber of such a library has a means for uniquely identifying the
`sequence of each oligomer. Methods for screening such
`libraries and reagents useful for their production are also
`provided.
`
`Glossary
`The folloWing terms are intended to have the folloWing
`general meanings as they are used herein:
`Complementary or substantially complementary: Refers
`to base pairing betWeen nucleotides or nucleic acids, such
`as, for instance, betWeen the tWo strands of a double
`stranded DNA molecule or betWeen an oligonucleotide
`primer and a primer binding site on a single stranded nucleic
`acid to be sequenced or ampli?ed. Complementary nucle
`otides are, generally, A and T (or A and U), or C and G. TWo
`single stranded RNA or DNA molecules are said to be
`substantially complementary When the nucleotides of one
`strand, optimally aligned and compared and With appropri
`ate nucleotide insertions or deletions, pair With at least about
`80% of the nucleotides of the other strand, usually at least
`about 90% to 95%, and more preferably at least about 98 to
`99.5%.
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6,143,497
`
`4
`Alternatively, substantial complementarity eXists When an
`RNA or DNA strand Will hybridiZe under selective hybrid
`iZation conditions to its complement. Typically, selective
`hybridiZation Will occur When there is at least about 55%
`identity over a stretch of at least 14 to 25 nucleotides,
`preferably at least about 65%, more preferably at least about
`75%, and most preferably at least about 90% identity. See,
`M. Kanehisa NucleicAcia's Res. 12:203 (1984), incorporated
`herein by reference.
`Stringent hybridiZation conditions Will typically include
`salt concentrations of less than about 1 M, more usually less
`than about 500 mM and preferably less than about 200 mM.
`The hybridiZation temperature for oligomers Will typically
`be greater than 22° C., more typically greater than about 30°
`C., and preferably in eXcess of about 37° C. Longer frag
`ments may require higher hybridiZation temperatures for
`speci?c hybridiZation. As other factors may dramatically
`affect the stringency of hybridiZation, including base com
`position and length of the complementary strands, presence
`of organic solvents and eXtent of base mismatching, the
`combination of parameters is more important than the abso
`lute measure of any one alone.
`Epitope: The portion of an antigen molecule Which is
`delineated by the area of interaction With the subclass of
`receptors knoWn as antibodies.
`Identi?er tag: A means Whereby one can identify Which
`monomer reactions an individual solid support has experi
`enced in the synthesis of an oligomer. The identi?er tag also
`records the step in the synthesis series in Which the solid
`support visited that monomer reaction. The identi?er tag
`may be any recogniZable feature Which is, for example:
`microscopically distinguishable in shape, siZe, color, optical
`density, etc.; differently absorbing or emitting of light;
`chemically reactive; magnetically or electronically encoded;
`or in some other Way distinctively marked With the required
`information, and decipherable at the level of one (or feW)
`solid support(s). A preferred eXample of such an identi?er
`tag is an oligonucleotide sequence.
`Ligand: A ligand is a molecule that is recogniZed by a
`particular receptor. The agent bound by or reacting With a
`receptor is called a “ligand”, a term Which is de?nitionally
`meaningful only in terms of its counterpart receptor. The
`term “ligand” does not imply any particular molecular siZe
`or other structural or compositional feature other than that
`the substance in question is capable of binding or otherWise
`interacting With the receptor. Also, a ligand may serve either
`as the natural ligand to Which the receptor binds, or as a
`functional analogue that may act as an agonist or antagonist.
`EXamples of ligands that can be investigated by this inven
`tion include, but are not restricted to, agonists and antago
`nists for cell membrane receptors, toXins and venoms, viral
`epitopes, hormones (e.g., opiates, steroids, etc.), hormone
`receptors, peptides, enZymes, enZyme substrates, cofactors,
`drugs, proteins, and monoclonal antibodies.
`Monomer: Any member of the set of molecules Which can
`be joined together to form an oligomer or polymer. The set
`of monomers useful in the present invention includes, but is
`not restricted to, for the eXample of peptide synthesis, the set
`of L-amino acids, D-amino acids, or synthetic amino acids.
`As used herein, monomers refers to any member of a basis
`set for synthesis of an oligomer. For example, dimers of
`L-amino acids form a basis set of 400 monomers for
`synthesis of polypeptides. Different basis sets of monomers
`may be used at successive steps in the synthesis of a
`polymer.
`Oligomer or Polymer: The oligomer or polymer
`sequences of the present invention are formed from the
`
`
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`6,143,497
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`5
`chemical or enzymatic addition of monomer subunits. Such
`oligomers include, for example, both linear, cyclic, and
`branched polymers of nucleic acids, polysaccharides,
`phospholipids, and peptides having either ot-, [3-, or uu-amino
`acids, heteropolymers in Which a knoWn drug is covalently
`bound to any of the above, polyurethanes, polyesters,
`polycarbonates, polyureas, polyamides, polyethyleneimines,
`polyarylene sul?des, polysiloxanes, polyimides,
`polyacetates, or other polymers Which Will be readily appar
`ent to one skilled in the art upon revieW of this disclosure.
`Peptide: Apeptide is an oligomer in Which the monomers
`are alpha amino acids and Which are joined together through
`amide bonds and alternatively referred to as a polypeptide.
`In the context of this speci?cation it should be appreciated
`that the amino acids may be the L-optical isomer or the
`D-optical isomer. Peptides are more than tWo amino acid
`monomers long, and often more than amino acid monomers
`long. Standard abbreviations for amino acids are used (e.g.,
`P for proline). These abbreviations are included in Stryer,
`Biochemistry, Third Ed., 1988, Which is incorporated herein
`by reference.
`Oligonucleotides: An oligonucleotide is a single-stranded
`DNA or RNA molecule, typically prepared by synthetic
`means. Those oligonucleotides employed in the present
`invention Will be 50 to 150 nucleotides in length, preferably
`from 80 to 120 nucleotides, and most preferably about 100
`nucleotides, although oligonucleotides of different length
`may be appropriate. Suitable oligonucleotides may be pre
`pared by the phosphoramidite method described by Beau
`cage and Carruthers, Tetra. Letts. 22:1859—1862 (1981), or
`by the triester method according to Matteucci, et al., J. Am.
`Chem. Soc., 103:3185 (1981), both incorporated herein by
`reference, or by other methods such as commercial auto
`mated oligonucleotide synthesiZers.
`Operably linked: A nucleic acid is operably linked When
`it is placed into a functional relationship With another
`nucleic acid sequence. For instance, a promoter or enhancer
`is operably linked to a coding sequence if it affects the
`transcription of the sequence. Generally, operably linked
`means that the DNA sequences being linked are contiguous
`and, Where necessary to join tWo protein coding regions,
`contiguous and in reading frame.
`Receptor: A molecule that has an af?nity for a given
`ligand. Receptors may be naturally-occurring or manmade
`molecules. Also, they can be employed in their unaltered
`natural or isolated state or as aggregates With other species.
`Receptors may be attached, covalently or noncovalently, to
`a binding member, either directly or via a speci?c binding
`substance. Examples of receptors Which can be employed by
`this invention include, but are not restricted to, antibodies,
`cell membrane receptors, monoclonal antibodies and antis
`era reactive With speci?c antigenic determinants (such as on
`viruses, cells or other materials), drugs, polynucleotides,
`nucleic acids, peptides, cofactors, lectins, sugars,
`polysaccharides, cells, cellular membranes, and organelles.
`Receptors are sometimes referred to in the art as anti
`ligands. As the term receptors is used herein, no difference
`in meaning is intended. A “ligand-receptor pair” is formed
`When tWo macromolecules have combined through molecu
`lar recognition to form a complex.
`Other examples of receptors Which can be investigated by
`this invention include but are not restricted to:
`a) Microorganism receptors: Determination of ligands
`that bind to receptors, such as speci?c transport pro
`teins or enZymes essential to survival of
`microorganisms, is useful in a neW class of antibiotics.
`
`15
`
`3O
`
`35
`
`45
`
`55
`
`65
`
`6
`Of particular value Would be antibiotics against oppor
`tunistic fungi, protoZoa, and those bacteria resistant to
`the antibiotics in current use.
`b) EnZymes: For instance, the binding site of enZymes
`such as the enZymes responsible for cleaving neu
`rotransmitters. Determination of ligands that bind to
`certain receptors, and thus modulate the action of the
`enZymes that cleave the different neurotransmitters, is
`useful in the development of drugs that can be used in
`the treatment of disorders of neurotransmission.
`c) Antibodies: For instance, the invention may be useful
`in investigating the ligand-binding site on the antibody
`molecule Which combines With the epitope of an anti
`gen of interest. Determining a sequence that mimics an
`antigenic epitope may lead to the development of
`vaccines of Which the immunogen is based on one or
`more of such sequences, or lead to the development of
`related diagnostic agents or compounds useful in thera
`peutic treatments such as for autoimmune diseases
`(e.g., by blocking the binding of the “self” antibodies).
`d) Nucleic Acids: The invention may be useful in inves
`tigating sequences of nucleic acids acting as binding
`sites for cellular proteins (“trans-acting factors”). Such
`sequences may include, e.g., enhancers or promoter
`sequences.
`e) Catalytic Polyveptides: Polymers, preferably
`polypeptides, Which are capable of promoting a chemi
`cal reaction involving the conversion of one or more
`reactants to one or more products. Such polypeptides
`generally include a binding site speci?c for at least one
`reactant or reaction intermediate and an active func
`tionality proximate to the binding site, Which function
`ality is capable of chemically modifying the bound
`reactant. Catalytic polypeptides are described in,
`Lerner, R. A., et al., Science 252: 659 (1991), Which is
`incorporated herein by reference.
`f) Hormone receptors: For instance, the receptors for
`insulin and groWth hormone. Determination of the
`ligands Which bind With high af?nity to a receptor is
`useful in the development of, for example, an oral
`replacement of the daily injections Which diabetics
`must take to relieve the symptoms of diabetes, and in
`the other case, a replacement for the scarce human
`groWth hormone that can only be obtained from cadav
`ers or by recombinant DNA technology. Other
`examples are the vasoconstrictive hormone receptors;
`determination of those ligands that bind to a receptor
`may lead to the development of drugs to control blood
`pressure.
`g) Opiate receptors: Determination of ligands that bind to
`the opiate receptors in the brain is useful in the devel
`opment of less-addictive replacements for morphine
`and related drugs.
`Substrate or Solid Support: A material having a rigid or
`semi-rigid surface. Such materials Will preferably take the
`form of small beads, pellets, disks or other convenient
`forms, although other forms may be used. In some
`embodiments, at least one surface of the substrate Will be
`substantially ?at. A roughly spherical shape is preferred.
`Synthetic: Produced by in vitro chemical or enZymatic
`synthesis. The synthetic libraries of the present invention
`may be contrasted With those in viral or plasmid vectors, for
`instance, Which may be propagated in bacterial, yeast, or
`other living hosts.
`Methods for Producing Large Synthetic Oligomer
`Libraries
`A general method of random oligomer synthesis is pro
`vided that produces the enormous numbers of compounds
`
`
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`6,143,497
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`8
`a) apportioning the supports among a plurality of reaction
`vessels;
`b) exposing the supports in each reaction vessel to a ?rst
`oligomer monomer and to a ?rst identi?er tag mono
`mer;
`c) pooling the supports;
`d) apportioning the supports among a plurality of reaction
`vessels; and
`e) exposing the supports to a second oligomer monomer
`and to a second identi?er tag monomer.
`In one embodiment, the steps of this process Will be
`repeated one to about 20 times.
`Alternatively to exposing the solid supports to a oligomer
`monomer and an identi?er tag monomer at the same time,
`the supports may be exposed sequentially to the ?rst oligo
`mer monomer and then to the ?rst identi?er tag monomer.
`The supports are then pooled and exposed to the second
`oligomer monomer and then to the second identi?er tag
`monomer. These steps are then repeated, typically from one
`to about times.
`The invention is described herein primarily With regard to
`the preparation of molecules containing sequences of amino
`acids, but could readily be applied in the preparation of other
`oligomers, as can be appreciated by those skilled in the art.
`There are various solid supports useful in preparation of
`the synthetic oligomer libraries of the present invention. It is
`understood that such solid supports are solid phase supports
`commonly used for solid phase synthesis of, for example,
`such oligomers as enumerated above, and thus are Well
`knoWn to those skilled in the art. In some embodiments of
`the present invention, such solid supports have novel fea
`tures as described beloW.
`The chemical or enZymatic synthesis of the oligomer
`libraries of the present invention takes place on such solid
`supports. The term “solid support” as used he