I,,, ... '�'"' II" "II''
`.l . , .I
`uL u .... ILli ,, .. h, .·
`
`"I' ''II
`I''! 1'1'''1
`
`071762. s�-·
`
`09/18/91/
`
`PCTIUS 9 2/ 0 7 8 1 5
`U.S.· PATE:\T ArPLICA T!O:\
`1802
`435
`
`WILLIAM J. DOWER, MENLO PARK, CA; RONALD W. BARRETT, SUNNYVALE, CA:
`
`MARK A. GALLOP, E. PALO ALTO, CA.
`
`**CONTINUING DATA*********************
`VERIFIED
`
`REC'D
`VII PO
`
`1 3 0 CT 1992
`PCT
`
`-
`
`.;
`
`I PRIORITY DOCUMENT I
`
`******
`**FOREIGN/PCT APPLICATIONS******
`' .. VERIF�ED
`
`'!'
`I ----
`
`I
`
`FOREIGN FILING LICENSE GRANTED 12/�6/91
`
`***** SMALL ENTITY *****
`
`DRAWING
`
`CL.AIMS
`
`CLAIMS
`
`CA
`
`9
`
`23
`
`7
`
`RECEIVED
`
`s 525.00 11509-36
`
`TOWNSEND & TOWNSEND
`STEUART ST. TOWER
`ONE MARKET PLAZA
`
`�
`�
`
`.., !
`' f.
`
`:_�
`
`. f
`-� �: .
`�:-J
`
`•
`
`. ..
`.. -.: .. . .. .
`�---� -· ..... �
`--�::��:-��---:�-:·:..�.": .. :_:.--.:..>�-__:_:.
`-'-�,�':--·-c • _ _:_._0._ --�--�-�'� --�-- ·:·-,, , . . , .. o,
`.-
`. · .' . ..
`.. .
`.
`-
`.
`. .
`. -
`·--·:. ,._ .. ....t-4.-�---·---..._._:.-· . ·-�-·-·�·-�:_�_:.__-�__:_� �:_:.a.:. ......... ·.-�· ...... -_..:_._-�--.!_:.;. ..
`... .. _ -- .. -41\
`....
`' ....
`
`Date
`
`.·
`
`.
`
`.
`
`-
`
`. .. �-. ...
`
`�
`
`.:;-�
`
`_::_:_·
`
`-��-
`
`--·
`
`-
`
`.
`
`•")
`
`SAN FRANCISCO, CA 94105 ----��.
`I'
`-, '-. \ \, \ \ --L. .. l
`
`...,.
`
`�atent and Trademark
`
`By authority of the
`CO*IS \I�� �OF- PATE�S.AND TRAOEMARI<S
`
`��R METHOD OF SYNTHESIZING DIVERSE COLLECTIONS OF OLIGOMERS
`Tnis is to certify tbat annexed hereto is a true copy from the recor<::s of tl'le United Stz.:e::
`Office of the application as originally f1led �n•cn is tcenttf•eo 3�c-�.
`. Certifying Officer
`1992
`
`I
`
`/ . '
`z.·'--'"--- --
`
`--·
`
`?
`;i· .-
`
`
`
`Page 1 of 60
`
`ILMN EXHIBIT 1009
`
`

`
`/
`
`; '!
`I
`
`'/
`
`I
`
`Page 2 of 60
`
`
`Page 2 of 60
`
`
`

`
`.
`
`
`.
`. . .
`..... -...
`
`·.
`
`.·
`
`�
`.
`.-_ .•
`
`. ':'-----..
`\ A�
`. · ... ·. -·
`.
`
`- # .;.... __
`'-·.-.· . ..,
`.·._.·
`_ ..
`•••
`(�
`
`PATENT APPLICATION SERIAL NO. -------
`
`U.S. DEPARTMENT OF CO�ERCE
`
`PATENT AND TRADEMARK
`
`OF:'I CE
`
`FEE RECORD SHEET
`
`· .
`
`i:>: . . C'_;'
`
`, -,l.?.i·.:
`
`1." \�� -l
`
`v?::.-.. """,�r
`
`':
`
`. ... ,,_ ":'-- _--..
`
`..._,.;,._
`.•--.
`J
`·�o_
`
`•
`
`. ..
`
`(5/87)
`PTO-ISSn
`..
`
`•
`
`· .. •-
`
`... .. : ...... . ·- � �
`
`•
`
`•
`
` J
`
`..
`
`-
`
`• .
`
`... ..
`
`...
`
`- _______ ... __ _
`
`•
`
`•
`
`....
`
`�-
`
`.Ill
`
`•
`
`_-__ .... ------·
`:..
`.
`..
`
`__ r.) __ . __ ·�--�-.
`
`.,
`
`•
`
`•
`
`•
`
`•
`
`. .. '! .... -.··-- --·-
`
`·
`
`· "'
`
`· - ·
`
`
`
`Page 3 of 60
`
`

`
`1-
`
`.---
`
`5 10
`15
`20
`25
`
`35
`
`--..
`
`. .
`
`� ...-::'' 'J.
`.. ')_
`
`A further a spect of the i nvention re lates to the
`
`stochast i c method for synthes iiing random o l igomers on
`part icles .
`use of ident i f ication tags on the part ic les to facil itate
`ident i f ication of the o l igomer s·equer'lce .
`
`BACKGROUND OF THE I NVENTION
`The relationship between structure and act ivity o f
`molecules i s a fundamenta l issue in the study of biolog ica l
`systems. Structure-activity relationships are important in
`understa nding , for example , the funct ion of enzymes , the ways
`i n which cel l s communicate wi th each other, as wel l a s cel lular
`control and feedback systems .
`Certa i n macromol�cules are known
`to· interact and bind to other molecules hav ing a very spec i f ic
`three-d imens ional spatial and electron i c distribution .
`Any
`large molecu l e hav i ng such specificity can be cons idered a
`receptor , whether i t i s an enzyme catalyz ing hydroly s i s of a
`metabo l i c i nt�rmediate , a cel l - surface protein med i a t i ng
`membrane transport of ions , a glycoprotein serving to ident i fy
`a particular cel l to its ne ighbors, an IgG-c1a�s ant i body
`
`c irculat i ng in the plasma , an ol igonucleotide sequence of DNA
`
`in the nucleus , or the l i ke. The various mol ecules which
`receptors sel ectively b i nd are known a s l igauds .
`Manr assays are ava i l able f or measuring the bind i ng
`
`affinity of known receptors a nd lig�nds , but the information
`
`Novel
`the number and type of l igands which are avai l able.
`ligands are sometimes d iscovered by chance or by app l icat ion of
`new techniques for the e l ucidat ion o f molecul ar structure,
`
`genetic techniqt1es for prote ins .
`
`Attorney Docket No . 11509-36
`METHOp OF SYNTHESIZING DIVERSE COLLECT IONS Of OLIGO�ERS
`The present invention relates general ly to a general
`. ----�----.-.--�----
`..
`
`1
`
`P.h.T�
`
`FIELD OF THE INVENTION
`
` ·.·
`.
`.
`:.::l
`�··.:.
`... · .... · ..
`'-:··.·-:-r�
`-. '.
`which caq be-gained from such experiments is o ften lirnitud by
`�-
`··� .. . .
`� �··
`-.�
`including x-ray crysta l l ographi c ana lysis and recombinant
`:,
`.
`• .:. ... .
`. ·· .. >
`-__ ,,_..:.;....._:.....: -r�·--· �,.. ..
` i-.�· _,1 __ -. L-., • -�
`---·•�·-�-• ._ .. _ .. f __
`' . -:-� �-. ·-�-.-:· -!-�· -'-; -�
`.
`.
`\
`..
`··6
`•
`. ...
`
`
`
`Page 4 of 60
`
`

`
`·-
`
`5
`
`10
`
`15
`
`20
`
`25
`
`JO
`
`35
`
`2
`
`Small peptides are an exemplary system for exploring
`
`the 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 polyme ric
`molecules they form a wide variety of three-dimensional
`configurations, each resulting from a particular amino ac id
`The number of possible
`sequence and solvent condition.
`pentapeptides of the 20 naturall y occurring amino acids, for
`example, is 205 or 3.2 million different peptides.
`The
`likelihood that molecules of this size might be useful in
`receptcr-binding studies is supported by epitopd analysis
`studies showing that some antibodies recognize sequences as
`short as a few amino acids with high �peci!icity. 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
`relationships.
`In most cases contemporary pharmaceutical
`research can be described as the process of discovering novel
`ligands with desirable patterns of specificity for biologically
`important receptors. Another example is research to discover
`new compounds for use in agriculture, such as pesticides and
`herbicides.
`Prior methods of preparing large numbers of different
`oligomers have been painstakingly slow when us�d at a scale
`sufficient to permit effective rational or random screening.
`
`(1963)
`For example, the "Merrifield" method (J. Am. Cher.1. Soc.
`85:2149-2154, _which is incorporated herein by reference) l:L1s
`I� the
`support made of an insoluble polymer. Another amino acid with
`
`been used to synthesize peptides on a solid support.
`Merrifield method, an amino acid is covalently bonded to a
`
`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
`
`·.
`·�-: .. ·
`. . ..
`;_
`-•-- -. -' ---__ , .., --•---oo:·-·--· ··-----·"- -• ·--- ' - . •... --
`:... . .. ..: ..
`\ ! .-'
`. \
`.··
`•
`. ..
`• •
`
`.
`
`- _.,.
`
`.
`
`•
`
`--·------
`·"'
`
`-·
`-� ---
`
`. ·}
`
`•
`
`•
`
`•
`
`0
`
`. .,
`
`. ·_ .
`
`
`::ti
`
`.
`
`.
`-
`
` �
`
`\.·-.. �
`---·--
`'--�--- �
`-�.::.----�
`'
`. .'· .·-.
`a
`I
`..
`r-
`t
`i-�---
`.
`(.
`.
`t�----,-'--
`f:..:.--. ... ......:.
`l· �
`'
`[
`.
`r· ,_..
`.
`
`
`
`Page 5 of 60
`
`

`
`p rotect ive group is added to the dipep t ide. This·process is
`practical to synthes ize more than a handful of peptide
`sequences in a day •
`For example , a tubul a r reactor system
`To synthes ize larger numbers of ol igomer sequences� ·
`
`it has also been proposed to use a ser i e s of reaction vessel$
`
`for oligomer synthesjs,
`
`cont i nued until a peptide of a desired length and sequence is
`
`obta ined. Us i ng the Merrifield method, i t i s not economically
`
`3
`
`may be used to synthes ize a l inear ol i gomer on a so l id pha se
`
`10
`
`support b y automated sequent i a l add i t i o n of reage nts .
`
`Th i s
`
`method s t i l l doe s not enabl e the syr.thesis o f a suffic ient l y
`
`.
`'."
`'.'
`.
`
`-�
`
`5
`
`15
`
`2 0
`
`25
`
`3 0
`
`3 5
`
`. : ....
`·-�-..
`J
`-
`·-····-··- ·--'·
`
`J
`
`screen ing.
`
`large nurobe r of ol igomer sequences for effect ive economica l
`
`Methods of prepar i ng a plura l ity of ol igome r
`sequences a re a l so known in which a forami nous container
`encloses a known quant ity of reactive solid supports , the so l id
`
`supports b e ing l a rger i n s ize than ope n i ngs of the container.
`
`The containers may be sel ectively reacted with des ired
`
`materi a l s to synthes ize des ired sequences of p roduct molecul es.
`
`As with other methods known in the art , th i s method c annot
`
`pract ica l ly be used to synthes ize a suff ic i e nt variety of
`
`pol ypept ides for e ffect ive screening .
`
`Othe r techniques have also been described .
`
`rhese
`
`methods include the synthes i s of pept ides on 96 p l as ti c p i ns
`
`which fit the format of standard microt it er p l ates.
`
`Unfortunate l y , whi l e these techn iques h ave been somewhat
`
`For examp l e , these
`useful, substantial prob l ems remain.
`methods continue to be l imi ted in the diversity of s equences
`
`which can be economical ly synthes ized a nd screened.
`
`From the ab?ve , it is seen that an improved method
`
`....... _· ··.::
`'I
`.·
`.-_
`
`and apparatus for synthes izing a diverse col l e�t ion of chemical
`sequences is des ired . Sl�Y OF THE INVENTION
`method for synthes izing random ol igomers on sol id supports, or
`monomers , the monomers being any member o f the set of mol ecules
`•
`•
`-•- -·�._.; .. __ .,_ ·-··
`... �
`,_ --.--�---=-· .•.
`.
`·)
`.· �
`
`The present i nv£ntion prov ides a gene ral stochastic
`
`p�ct icles . The ol igomers .are composed o f a sequence of
`
`•
`
`•
`
`•
`
`
`
`Page 6 of 60
`
`

`
`4
`large l ibrary of sol id supports , each support havi ng attach�d a
`
`i. e. ami no acids, nucleic acids, carbohydrates, lipids,
`
`polyesters , and the l ike . The method involves producing a
`
`which can be joined together to form an ol igomer or poly::1er,
`
`s ing l e ol igomer s equence , the oligomers being synthesized in a
`
`random comb inatorial
`
`( "stochas t ic") fash i on. The library is
`
`then screened to i solate individu a l solid supports carrying
`
`ol igomers that b i nd to a receptor.
`
`Each ol igornr sequence in
`
`the l ibrary is un iqu e , in a preferred e�bodirnent.
`
`In another
`
`preferred embodiment, the sol id supports are nonporous beads.
`
`The sol id supports may be composed of a s ingl e part i cl e , or two
`
`or more l inked parti cles.
`
`A further embodiment of the i nvent ion is the use of
`
`an ident ifier tag t o ident ify the sequence of monomers in the
`
`ol igomer . The ident ifier tag , which may be attached to the
`
`same part i c l e as the oligomer or to a second part i c l e attached
`
`:--..
`
`to the ol igomer-carrying part icle, may be any recog�izabl e
`
`feature that in scme way carries the requ ired information , and
`
`that i s dec i pherab l e at the l eve l of cne or a few sol id
`
`support s. The sol id supports may be joined to the ol igomers
`and the ident ifier tag by means of a l iriker molecul e.
`I n a preferred embodiment, the ident if ier tag w i l l b e
`
`an ol igonuc l eotide , preferabl y compose d of pyrimidi nes . The
`
`ampl if i cat ion site, to a l low amp l ificat i on of the t a g by , for
`
`exampl e, po l ymerase chain reaction . A DNA sequencing primer
`
`s i te , which may be spec ific for each step of the ol igomer
`
`synthe s i s, may a l so be included in the ol igonucleot ide tag .
`
`The tag may be des igned to include , in the ol igonucl eot i de
`
`sequence, i nformat ion al lowing ident ification of the monomer
`
`assoc iated with the addit ion of the particular tag .
`
`The
`
`preferred em.,odiment .
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`ol igomer synthe s i s on particl es.
`
`ol igonucl eot ide identifier tag �ay conta i n a 5' and a 3 '
`ol igonucleot id� w i l l be about 100 nucleotides in length, i� a
`F i g . 1 i s a schemat ic representat ion of combinatorial
`.....
`....
`
`-· ·.····•·
`
`•
`
`•
`
`•
`
`•
`
`.
`
`•
`
`e
`
`ca
`
`!'t
`
`5
`15
`
`10
`
`20
`
`25
`
`3 0
`
`··--.: i
`) -·
`-� .... _J
`
`.·.·e·
`
`�-� :�
`- (-)
`
`3 5
`
`---}
`---···-···-··.
`
`•
`
`
`
`Page 7 of 60
`
`

`
`- .. . . -
`.,�-
`
` J
`
`
`
`. .
`
`.,
`
`5
`
`Fig . 2 is a schematic repres�ntat ion o f concur rent
`combinator i a l ol igome r synthesis and particle tagging.
`Fig . 3 is a description of: one method o f bead
`functiona liz ation , the compat ib l e chemistr ies f o r peptide
`synthesis and round by round a ttachment o f oligonucleot ide
`identi f i er tags , incl uding synthesis of a m i no- functiona l ized
`beads , the structure o f p rotected 5' ma l e imidyl
`ol i gonucleotides , ami no acid coupling and introduct ion o f a
`thio l "handle , " step-specif i c oligonucl eotide attachment to a
`
`bead , subsequent amino acid coupling (s) and oJ. igonucleotide
`attachment ( s) , and peptide and ol igonucleot ide ueprotect i on.
`
`Fig. 4 is a schemat i c representat ion o f one example
`
`·of an oligonucleotide tag.·
`
`DESCRIPTION OF THE SPECIFIC EMBODIMENTS
`
`The present invention prov ides novel methods and
`instru�ents for producing large synthetic oligomer librar ies.
`
`In a pre ferred embodiment of the present invention, each member
`of such a library has a means for un ique l y identifying the
`
`sequence o f each oligcmer. · Methods for screening such
`libraries and reagents useful for the ir producti on a re a l so
`p rov ided.
`
`Glossary
`
`Re fers
`
`The follow ing te rms are intended to have the
`following general meanings as they are used herein:
`
`complementary or substa ntially complementary:
`to base pairing between nucleotides or nucleic acids , such as,
`for instance , betwee n the two stra nds o f a double stranded DNA
`nol ecule or between an oligonucleotide primer and a primer
`bind i ng site on a singl e stranded nucleic acid to be seque nced
`.Comple::nentary nucleotides are, generally, A and
`or amp:l ifie
`d.
`( or A and U), or c and G. Two sing l e stranded RNA or DNA
`_
`T
`mol ecules are sa id to be substantia l l y complementary when the
`nucleotides of one strand , optimally aligned and compared and
`with appropriate nucleotide insertions or Je letions, pai r w i th
`�t least abou� 80% o f the nucleotides o f the other strand ,
`
`-· .
`. ·
`
`.·
`
`��
`
`...
`1 ...... ••
`....J.
`
`' .. * T
`�
`
`'
`
`•
`
`10
`
`20
`
`25
`
`5
`
`10
`
`-.. . � .. . .
`--·.····
`
`··--·- .. - - -- .
`•
`• ••
`•
`- 11 -
`• • � • � ... ... • "'
`• • • • •
`•
`. ..
`.
`'
`..
`
`.. ,_. .. -�
`
`··�
`
`.
`
`·- .
`
`•
`
`•
`
`•
`
`•
`
`
`
`Page 8 of 60
`
`

`
`_..._,.
`
`6
`
`usually at least about 90\ to 9�%, and more preferably at least
`
`about 98 to 99.5%.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`-·--
`.
`. .
`
`.
`
`. ..
`
`.
`
`. �
`
`.
`
`.
`·'
`. -. ..
`.• J
`------:-· _ ... _
`
`•
`
`'
`
`.
`
`.
`
`.
`
`.
`
`.
`• .
`
`•
`
`'
`
`•
`
`•
`
`•
`
`•
`
`•
`
`•
`
`•
`
`••
`
`..
`
`.
`
`.
`
`.
`..
`
`.
`
`
`
`•
`
`•
`
`Alternatively, substantial complementarity exists
`when an RNA or DNA strand will hybridize under selective
`hybrldization conditions �o its complement. Typically,
`selective hybridization will occur when there is at least about
`55\ identity over a stretch of at lea�t 14 to 25 nucleotide3,
`preferably at least about 65%, more �referably at least about.
`75�, and most prefer�bly at least about 90% identity.
`Kanehisa Nucleic Acids 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 hybridlzation temperature for oligomers will
`
`Se�, M.
`
`typic�lly be greater than 22•c, more typically greater than
`about Jo•c, and preferably in excess of about 37•c. Longer
`fragments may require higher hybridization temperatures for
`specific hybridization. As other factors may dramatically
`affect the stringency of hybridization, including base
`com,osition and length of the complementary strands, presence
`of organic solvents and extent of base mismatching, the
`combination of parameters i� more important than the absolute
`measure of any one alone.
`Epitope: The portion of an antigen molecule which is
`delineated by the area of interaction with the subcl�ss of
`receptors known as antibodies .
`�dentifier tag:
`
`A means whereby one cen identify
`
`which monomer reactions an individual solid support has
`experienced in the synthesis of an oligomer. The identifier
`
`) . -.
`
`·-------"'
`
`tag also records the step in the synthesis series in which the
`
`tag may be any recognizatle feature which is,
`for examt>le:
`microscopically disti�guishable in shape, size, color, optical
`density, etc.; differently absorbing or emitting of light;
`chemically reactive; magnetically or electronically encoded; or
`
`information, and decipherable at the level of one (or few)
`
`solid support visited that monomer reacti0n.
`The identifier
`in some other way dis�inctively marked with the required
`� ...... .
`. ..... -. . ..
`. .. . ......
`--.--� .. �·!.-.__ __ �'
`-· .. .
`. .. ·.: ...... -.-�-::--.". -�r-.-.-., -.�:.-.':.--�··-:·. ·: .• ... -· .•· •.
`-
`.
`.
`. .
`.
` ·
`·-.
`..
` • •.
`. •
`'
`--- •
`'
`.:
`··•
`#
`.·,
`. .,
`
`
`
`Page 9 of 60
`
`

`
`.. :�
`
`a particular receptor. The aqent·Dound by or reactinq with a
`
`The term
`
`7
`solid support(s). A preferred example ot such an identifier
`taq is an oliqonucleoti�e sequence.
`Ligand: A ligand is a molecule that is. recognized by
`receptor ia called a "liqand", a term which is definitionally
`meaningful only in terms of it$ counterpart receptor.
`"ligancl'" does net imply any particular molecular size or other
`structural or compositional teatura other than that the
`substance in ques�ion is capable of bindinq or otherwise
`interacting with the receptor. Also, a ligand may serve either
`�s the natural liqand. to which the receptor Dinds, or as a
`f�nct1onal analogue that may act as an agonist or antagonist.
`Examples of ligands that can be investigated by this invention
`incl�de, out are not restricted to, aqonists and antagonists
`for cell membrane receptors, toxins and venoms, viral epitopes,
`hormones (e.g., opiates, steroids, etc.), hormone receptors,
`paptidea, enzymes, enzyme substrates, cotac�ors, drugs,
`Monomer; Any member ot the se� of molecules which
`can be joined toqether to form an oligomer or polymer. The set
`ot monomers useful in the present invention includes, but. is
`not res�ricte4 to, fer the example of peptide synthesis, the
`set of L-amino acids, D•amino acids, or synthetic amino acids •
`As uaed herein, aonomers re!ers to any member of a basis set
`for synthesis o! an oligomer. For exa�ple, dimers of L-amino
`acicl5 form a basis set of 400 monomers for synthesis of
`polypeptides. Different basis sets of monomers �ay be used at
`successive steps in the synthesis of a polymer.
`Oligomer or polymer: The oligomer or polymer
`sequences ot-the present invention are formed from the chemical
`or enzymatic addition of monomer suDunits. such oliqomers
`incl�de, for example, both linear, eyclic, and bran�hed
`polymers of nucl eic acids, polysaccharides, phospholipids, and
`peptides havinq either 4-, �-. or w-amino acids, heteropolymers
`in which a known druq is covalently bound to any of the above,
`polyamides, polyethyleneimines, polyarylene sulfides,
`polyailoxanea, polyimidae, polyacetates, or other polymers
`
`proteins, and monoclonal antibodies.
`
`polyurethanes, polyesters, polycarbonatas, polyureas,
`
`•.
`....
`
`: ...
`il
`... . __.
`
`....
`
`5
`
`lO
`
`20
`
`JU
`
`J�
`
`
`
`Page 10 of 60
`
`

`
`a
`which will be readily apparent to one skilled in the art upon
`reviev of this disclosure.
`Peptide: A peptide is an oligomer in which the
`
`monomers are al pha amino acids and which are joined toqether.
`through amide bonds and alternatively referred to as a
`In the context of this specification it should be
`
`polypeptide.
`
`the D-optical isomer.
`mono�ers lonq, and often more than 20 amino acid monomers lonq.
`
`Oliqonycleotides: An oligonucleotide is a single-
`
`means.
`
`nucleo1:1des, al�llouqh ol1qonueleot1des of different lenqth may
`be appropriate.
`the pDosphora�niaite method descri))eci by Beauc:ac;e and
`
`appreciated that the amino acids may be the L-optical isomer or
`Peptides are more than two amino acid
`Standard abbreviations for amino acids are used (e.g., P for
`proline). These abbreviations are included in Stryer,
`8iocDemistry, Third Ed., 1988, which is incorporated herein by
`reference.
`stranded DNA or RNA �olecule , typically prepared by synthetic
`Those oligonucleotides employed in the present
`invention will be so to 150 nucleotides in length, preferably
`rrom·-so to 120 nucleotides, anc1 mos� preferably about 100
`Suitable oligonucleotides may be prepared by
`Carruthers, Tetra. L9tts. 22:1859•1862 (1981), or by the
`triester method according to Matteucci, et al., J. Am· c� ·
`�. lOJ:Jl85 (1911), both incorporated herein by reference,
`or by other methods such as commercial automated
`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
`opercbly linke4 to a coding sequence if it affects the
`means that the DNA sequences being linked are contiquous and,
`and in reading trame.
`Receptor: A molecule that has an affinity for a
`Also, they can be employed in their unaltered
`natural or isolated state or as aqqregates with other species.
`
`transcription of the sequence.
`
`Generally, operably linked
`
`�hera necessAry·to join two protein codinc; req ions , contiquous
`
`given 11qand. Receptors may be naturally-occurring or manmade
`molecules.
`
`....,
`
`i
`
`,
`
`5
`
`10
`
`15
`
`20
`
`25
`
`JU
`
`35
`
`
`
`Page 11 of 60
`
`

`
`��
`
`,_
`
`. �:
`� ...... · .....
`·�
`·--: -:--,•
`. ·
`.·
`.
`
`..
`.·
`·'·-�·-·
`
`;.· -.•' ... -
`! i
`� _J.. ·-•
`\
`� ...
`1-
`
`)-::
`
`•
`•
`
`...
`
`·:·
`.
`'
`----
`
`J
`
`5 10 15 20 25 30 35
`
`• •
`
`-
`
`9
`
`Receptors may be attach�d , coval ently o r noncova l e nt l y , to a
`bind i ng member , e i ther d i rectly or v i a a spe c i f i c bin d i ng
`substance .
`Examp l es o f receptors wh ich can be emp loyed by this
`invention incl ude , but are not restricted to , ant ibod i es ,
`ce l l memb rane receptors , monoclonal antibod ies and ant isera
`reactive with spec i f i c antigenic determinants (such aG on
`v i ru ses , cel l s .or other material s ) , drugs , polynuc l eo t ides ,
`n�c l e ic ac ids , pept ides , cofactors , lectins, sugars ,
`polysacchar ides , c e l l s , c e l l u l a r memb ranes , and organe lles.
`Rec eptors are somet imes re ferred tc i n the art as ant i - ligands.
`
`is intended . A " l i gand- receptor pai r " is formed when two
`
`form a comp l e x .
`Other e�ampl es o f receptors wh ich can be i nvest igated
`by th i s invent ion inc l ude but are not restr icted to:
`a )
`Microorganism rec eptor�:
`Determ i nation o f l igands that
`b i nd to receptors , such as spec i f ic transport proteins or
`
`i n a new c l ass o f ant ibiotics. Of part icul a r v a lue would
`be ant i b i otics aga inst opportuni s t i c fung i , protozoa , ·and
`those bacter i a res istant to the antibiot i cs i n cu rrent
`use .
`For instance , the bindi ng site o f enzymes such
`Enzymes :
`as the enzymes respons ib l e for c l e avi ng neurot r a nsmi tt ers .
`Determinat ion of ligands that b i nd to certa i n r eceptors ,
`
`the d i f ferent neurotransmitters, i s use ful i n the
`
`disorders o f neurotransmis�icn .
`
`i nv�stiqa t i ng the l igand-binding site on the antibody
`
`As the term rec eptors is used here in, no d i f f erence in mean ing
`macromol ecules have combined through mo l ecular recogn i t i on to
`enzymes essential to surv iva l of m icroorganisms , is u s eful
`and thus modu l ate the act ion of the enzymes that cleave
`�ntibodies:
`i�
`epitope may l ead to the deve lopment of va�cines of which
`;'I -
`. � -
`.
`
`devel opme�� o f drugs that can be used i n the t r e a tment of
`For instance, the invention m�y �e u�c�ul
`mol ecul e which comb ines wi th the ep itope of an a ntigen of
`' • •
`·.
`. --.. ---·-·--
`•
`•
`•
`•
`•
`• •
`•
`•
`•
`•
`
`b)
`
`c)
`
`• • .
`
`i nterest .
`
`Determi n i ng a sequence that mimi cs. an
`
`antigenic
`
`_
`
`the immunogen is based on one or more of such sequenc es,
`or �ead to the development of rel.:lted di agnost ic agen ts or
`compounds use ful i n therapeutic tre atments such as fo r
`
`..
`
`-
`
`;)
`
`.,
`
`"I
`
`
`
`Page 12 of 60
`
`

`
`��
`. .
`
`d)
`
`e)
`
`10
`
`. . �.· . : · ..
`
`l.,
`
`5
`... . � . .
`15 20 25
`· . .. . · . .. ·
`l .... ...
`f) g)
`35
`- �· ---· •-.
`
`:-ia:_·
`. ·� .
`
`30
`
`. ...
`
`·-·
`
`•. ---- .
`
`.
`
`.
`
`-
`
`-·-
`
`.
`
`.
`
`herein by reference.
`tLormone re�eptors: For instance, the receptors for
`insulin and growth hormone. Det�rmination of the ligands
`which bind with high affinity to a receptor is useful in
`the development of, for example, an oral rep:acement of
`the daily injectlons 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 cadavers or oy recombinant DNA
`technology. Other examples are the vasoconstrictive
`hormone receptors; determination of those ligands that
`bind to a receptor may lead to the devel�pment of drugs to
`control blood pressure.
`Opiate receptors: Determination of ligands that bi�d to
`the-op1ate receptors in the brain is useful in the
`development 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 so�e
`
`-------
`---------------
`
`10
`
`autoimmune diseases (e.g., by blocking the binding of the
`
`"self" antibodies) .
`Nucleic Acids: The invention may be useful in
`investigating sequences of nucleic acids acting as hinding
`sites for cellular prott3ins ("trans-acting factors") .
`such sequences may include, e.g., enhancers or promoter
`sequences.
`Cataly�ic Polypeptides: Polymers, preferably
`polypeptides, which are capable of promoting a chemical
`reaction involving the conversion of one or more reactants
`to one or more products. Such polypeptides generally
`include a binding site sp�cific for at least one reactant
`or reaction intermediate and an active functionality
`proximate to the binding site, which functionality is
`capable of chemically modifying the bound reactant.
`Catalytic polypeptides are describ�d in, Lerner, R.A., et
`
`al., Scienc� 252: 659 (199�). which is incorporated
`
`•
`
`·.
`
` �
`
`. � .
`
`. . .
`
`. .. •.
`
`•
`
`•
`
`_It_
`
`• ---"'-
`
`___ :·."�-•
`
`•
`
`•
`
`•
`
`• •
`
`•••
`
`•
`
`•
`
`•
`
`•
`
`
`
`Page 13 of 60
`
`

`
`11
`
`e mbodiments , at L�ast one su rface of t he subst rate wil l be
`subst ant ially f l�t.
`A rou ghl y sp he ric al s hape is p re f erred .
`Produced b y in vit ro c hem ic al or
`synt het ic:
`en zymat ic s ynt hes is .
`The s ynt hetic libr aries of t he present
`in vent ion may be contr asted w it h those in viral o r �l �sm id
`vecto rs , fo r inst ance , w hic h m ay be p rop agated in bacte rial,
`ye ast , o r ot he r li vin g hosts .
`
`Methods for P roduc in g Large synt het ic Oli go mer Lib ra ries
`A gener al met hod o f r an dom ol i go mer s ynt he s is i s
`p ro vided t hat produces t he eno rmo us nu mbers o f compounds
`availab le w ith recomb in ant s ystems and t he monome r set
`
`- di versit y av ail abl e wit n c hem ic al s yn t hesis met hods . By me ans
`
`o f t he p resent met hod it is poss ibl e to re ad ily p roduce up to
`
`1012 diffe ren t o ligo me rs , a d ramat ic imp ro ve ment ove r p re vious
`( n x = up to 108 to
`1012 compo un ds ).
`In one embod ime nt , n wil l be 5 o r 6, and t he
`co llecti on w ill cont ain about 105 o r 106 d i f fe rent membe rs ,
`
`It also p rov ides a f ac ile me ans o f o ligo me r
`me �hods .
`iden tif i cation . The gene ral met hod c o mp ri ses :
`(a) P roduc in g a lar ge , hig hly d i verse co l!ection or
`l ib rary, e ac h me mbe r o f suc h a l ib rary co mp ris in g s o lid
`suppo rts havin g att ac hed a sing le o lig.ome :t· sequence
`( e . g. , a
`pep ti de ) . The so lid suppo rt is at tac hed to t he o ligome r b y
`me ans of a linke r t hat has an app rop ri ate func ti on al group at
`e ach end , one group approp ri ate for att ac hment to t he suppo rt
`and t he ot he r group approp riate fo r att ac hment to the o l igome r.
`Such a co llect ion m ay cont ain , fo r ex amp le , al l com bin at i ons of
`n mono mers asse mb led into X len gt h o ligo mers
`
`(�
`
`. ..
`.··
`'
`'
`.
`.
`
`. '
`--, .. !
`.•
`
`respect i ve ly.
`I t m ay al so cont ain o l igo me rs havi n g d if f e rent
`mono mer units at , fo r ex ample , on ly one o r a sm all number of
`
`5 10
`
`15
`
`20
`
`25 )0
`
`.
`
`�-,--�
`
`.. " � . :
`.. '
`·.· .
`· .... · ..
`·•• -....
`-· ·-·
`•, .. ·."
`-·
`. •
`t
`.· .. --...:...:..
`-.""·� .. :.
`.... _-.'
`...
`.
`\ <�·-<·>:·
`..
`-... ' ...
`...
`_ ..
`,"
`' ... �.-�
`...
`..
`•
`• -. •
`.
`] ..
`J t *
`
`35
`
`-� .. � . •
`
`.
`
`•
`
`•
`
`I I i I
`
`pos itions w hi l e having an identical s e quence at all other
`( " stoc hastic ") f as hion b y· c he mic al and/o r enz ymat ic asse !T'.b l y of
`supports c arryi n g o li go mers active i n bind in g to a re cept or •
`. . . - -•
`"'
`--"l
`•
`•
`0
`•••
`•
`.
`.
`.
`' ------�-.�� �
`.,
`,,
`
`pos itions !
`(b) synt hes iz in g t he o li gome rs in a random comb in at ori al
`
`mono mer units :
`( c ) Screen in g't he co llect ion to iso l ate ind iv idu al s o l id
`
`•
`
`. •
`
`•
`
`�
`
`�
`
`•
`
`•
`
`••
`
`•
`
`•
`
`•
`
`•
`
`
`
`Page 14 of 60
`
`

`
`12
`A _synthetic oligomer library may be produced by
`synthesizing on each of a plurality cf solid supports a single
`
`oligo111er sequence, the oligomer sequence being different for
`
`different solid supports.
`
`The oligomer sequence is synthesized
`
`in a process comprising th� steps of:
`
`a)
`
`apportioning the supports in a stochastic manner
`
`b)
`
`C)
`
`d)
`
`among a plurality of reaction vessels;
`
`exposing the supports in each reaction vessel to
`
`a f}rst monomer;
`
`pooling the supports;
`
`apportioning the supports in a stochastic manner
`
`among the plurality of reaction vessels;
`
`--�-
`
`e)
`
`exposing the supports in each reaction vessel to
`
`a seco·nd monomer; and
`
`repeating steps n) through e) from at least one
`
`f)
`to ·twenty times.
`Typically, substantially eq-oJal numbers of solid
`
`supports will be apportioned to each redction vessel.
`
`In onP.
`
`embodi�ent of the method, the monomers are chosen from the set
`
`of amino acids and the resulting oligomer is a peptide.
`In a preferred embodiment of the invention, the solid
`supports on which the oligom�rs are synthesized also have
`attached an identifier tag that can be easily decoded to report
`
`5
`
`10
`
`15
`
`20
`
`
`
`.... � . _, .
`--3
`:- _ ....
`..
`
`,-..... :....··/
`. . . ... ,
`·-.
`
`-)
`
`.
`
`.
`
`25
`
`the sequence of the oligomer contained on each solid support.
`
`The identifier tags may be attached to the solid support by
`
`means of a linker that has an appropriate functional group at
`
`each end, one group appropriate for attachment to the support
`
`and the other group appropriate for attachment to the
`
`30
`
`35
`
`identifier tag.
`
`It will be readily appreciated after reading
`
`the disclosure below that one could also produce large
`synthetic oligomer lioraries lacking identifier tags.
`_A synthetic oligomer library that incorpor�tcs
`i�entifiP-r tags is produced by synthesizing on each of a
`plurality of solid supports a single oligomer sequence and one
`or more identifier tags identifying the oligomer sequence. The
`oligomer sequence and identifier tags are synthesized in a
`
`process comprising the steps of:
`
`'-� ....
`- ...... · ...
`
`) ?·
`·. --..:..:.
`� - .
`.
`.. ·_. - .
`
`..
`
`•
`
`•. � - '·-
`
`...
`.
`.. .
`.. - �
`
`. �- - .-•-·- - . - ·1 ... - ..
`
`'
`
`f.--
`
`•
`
`•
`
`•
`
`• •
`
`··•
`
`•
`
`.
`
`- .
`
`•
`
`
`
`Page 15 of 60
`
`

`
`13
`
`apportioning the supports among a p�u ral ity of
`
`react ion vesse l s:
`
`expos ing the supports in each reaction vessel to
`apportioning the supports among a p l ura l i ty o f
`
`a f i rst ol igomer monomer