PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 5 :
`(11) International Publication Number:
`WO 93/09668
`AOIN 1/02, C12Q 1/00
`GOIN 33/566, 33/543, BOIS 19/00
`C07D 471/02, 235/00, 473/00
`C07D 235/30, C07K 1/04
`CO7K 17/06, 17/14
`
`
`
`
`
`Al
`
`(43) International Publication Date:
`
`27 May 1993 (27.05.93)
`
`
`
`nd
`
`a PCT/US92/10183|(72) Inventors; and(21) International Application Number:
`
`(75) Inventors/Applicants (for US only) : WINKLER,James,L.
`
`(22) International Filing Date:
`20 November 1992 (20.11.92)
`[US/US]; 2140 Ash Street, Palo Alto, CA 94306 (US).
`FODOR,Stephan, P., A. [US/US]; 3863 Nathan Way,
`
`(30) Priority data:
`Palo Alto, CA 94303 (US), BUCHKO, Christopher, J.
`22 November1991 (22.11.91) US
`07/796,243
`[US/US]; 2222 Fuller Road, #405A, Ann Arbor, MI
`24 April 1992 (24.04.92)
`US
`07/874,849
`48105 (US). ROSS, Debra, A. [US/US]; 3419 Bridge-
`wood Terrace, #302, Fremont, CA 94536 (US). ALD-
`
`(60) Parent Applications or Grants
`WIN, Lois [US/US]; 179 Lakeshore Drive, San Mateo,
`(63) Related by Continuation
`CA 94402 (US). MODLIN,Douglas, N. [US/US]; 4063
`
`US
`Scripps Avenue, Palo Alto, CA 94306 (US).
`Filed on
`US
`Filed on
`
`
`07/796,243 (CIP)
`22 November1991 (22.11.91)
`07/874,849 (CIP)|
`24 April 1992 (24.04.92)
`
`(74) Agents: WEAVER,Jeffrey, K. et al.; Townsend and Town-
`send, One Market Plaza, 20th FI. Steuart Tower, San
`Francisco, CA 94105 (US).
`
`(71) Applicant (for ail designated States except US): AFFYMAX
`TECHNOLOGY N.V. [NL/NL]; De Ruyderkade 62,|
`Curacao (AN).
`
`(81) Designated States: AU, CA, JP, US, European patent (AT,
`BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC,
`NL, SE).
`
`Published
`With international search report.
`
`(54) Title: COMBINATORIAL STRATEGIES FOR POLYMER SYNTHESIS
`
`
`
` 413
`
`
`Er
`TIQU
`
`
`
`
`
`(57) Abstract
`
`
`
`
`
`
`
`
`A method and device for forming large arrays of polymers on a substrate (401). According to a preferred aspect of the in-
`vention, the substrate is contacted by a channel block (407) having channels (409) therein. Selected reagents are delivered through
`the channels, the substrate is rotated by a rotating stage (403), and the process is repeated to form arrays of polymers on the sub-
`strate. The method may be combined with light-directed methodologies.
`
`
`
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`Viet Nam
`
`France
`Gabon
`United. Kingdom
`Guinca
`Greece
`Hunpary
`(reland
`Italy
`Japan
`Democratic People’s Republic
`of Korca.
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Luacmbourg
`Monuco
`Madiigascar
`Mali
`Mongolia
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Braszit
`Canada
`Central African Republic
`Cango
`Switverland
`Céte dIvoire
`Cameroon
`Crechoslovakta
`Czech Republic
`Germany
`Denmark
`Spain
`Finfand
`
`Mauritania.
`Malawi
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovak Republic
`Senegal
`Sovict Union
`Chad
`Togo
`Ukraine
`United States of Amcrica
`
`

`

`WO 93/09668
`
`PCT/US92/10183
`
`COMBINATORIAL STRATEGIES FOR POLYMER SYNTHESIS
`
`BACKGROUND OF THE INVENTION
`
`This application is related to U.S. Serial No. 796,243
`(filed November 22, 1991) and to U.S. Serial No. 874,849 (filed April
`24, 1992), both of which are incorporated herein by reference for all
`purposes.
`
`The present invention relates to the field of polymer
`synthesis and screening. More specifically,
`in one embodiment the
`invention provides an improved method and system for synthesizing
`arrays of diverse polymer sequences. According to a specific aspect
`of the invention, a method of synthesizing diverse polymer sequences
`such as peptides or oligonucleotides is provided.
`The diverse
`polymer sequences may be used, for example,
`in screening studies for
`determination of binding affinity.
`Methods of synthesizing desired polymer sequences such as
`peptide sequences are well known to those of skill in the art.
`Methods of synthesizing oligonucleotides are found in, for example,
`Oligonucleotide Synthesis:
`A Practical Approach, Gate, ed.,
`IRL
`Press, Oxford (1984),
`incorporated herein by reference in its
`entirety for all purposes.
`The so-called "Merrifield" solid phase
`peptide synthesis has been in common use for several years and is
`described in Merrifield, J. Am. Chem. Soc.
`(1963) 85:2149-2154,
`incorporated herein by reference for all purposes. Solid-phase
`synthesis techniques have been provided for the synthesis of several
`peptide sequences on, for example, a number of "pins." See e.g,
`Geysen et al., J.
`Immun. Meth.
`(1987) 102:259-274,
`incorporated
`herein by reference for all purposes. Other solid-phase techniques
`involve, for example, synthesis of various peptide sequences on
`different cellulose disks supported in a column.
`See Frank and
`Doring, Tetrahedron (1988) 44:6031-6040,
`incorporated herein by
`reference for all purposes. Still other solid-phase techniques are
`described in U.S. Patent No. 4,728,502 issued to Hamill and Wo
`90/00626 (Beattie,
`inventor).
`Each of the above techniques produces only a relatively
`low density array of polymers.
`For example,
`the technique described
`in Geysen et al.
`is limited to producing 96 different polymers on
`pins spaced in the dimensions of a standard microtiter plate.
`Improved methods of forming large arrays of peptides,
`oligonucleotides, and other polymer sequences in a short period of
`time have been devised. Of particular note, Pirrung et al., U.S.
`Patent No. 5,143,854 (see also PCT Application No. WO 90/15070) ana
`Fodor et al., PCT Publication No. WO 92/10092, all incorporated
`herein by reference, disclose methods of forming vast arrays of
`peptides and other polymer sequences using, for example,
`light-—
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`2
`
`See also, Fodor et al., Science
`directed synthesis techniques.
`(1991) 251:767-777, also incorporated herein by reference for all
`purposes.
`
`Some work has been done to automate synthesis cf polymer
`arrays. For example, Southern, PCT Application No. WO 89 /10977
`describes the use of a conventional pen plotter to deposit three
`different monomers at twelve distinct locations on a substrate.
`These monomers were subsequently reacted to form three different
`polymers, each twelve monomers in length.
`The Southern Application
`also mentions the possibility of using an ink-jet printer to deposit
`monomers on a substrate. Further,
`in the above-referenced Fodor et
`al., PCT application, an elegant method is described for using a
`computer-controlled system to direct a VLSIPS™ procedure. Using this
`approach, one heterogenous array of polymers is converted, through
`simultaneous coupling at a number of reaction sites,
`into a different
`heterogenous array. This approach is referred to generally as a
`“combinatorial” synthesis.
`The VLSIPS™ techniques have met with substantial success.
`in some cases it is desirable to have alternate/additional
`However,
`methods of forming polymer sequences which would not utilize, for
`example, light as an activator, or which would not utilize light
`exclusively.
`
`SUMMARY OF THE INVENTION
`
`Methods and devices for synthesizing high-density arrays
`of diverse polymer sequences such as diverse peptides and
`oligonucleotides are provided by virtue of the present invention.
`addition, methods and devices for delivering (and, in some cases,
`immobilizing) available libraries of compounds on specific regions of
`a substrate are provided by this invention.
`In preferred
`embodiments, various monomers or other reactants are delivered to
`multiple reaction sites on a single substrate where they are reacted
`in parallel.
`
`In
`
`According to a preferred embodiment of the invention, a
`series of channels, grooves, or spots are formed on or adjacent a
`substrate. Reagents are selectively flowed through or deposited in
`the channels, grooves, or spots, forming an array having different
`compounds ~ and in some embodiments, classes of compounds - at
`selected locations on the substrate.
`
`According to the first specific aspect of the invention,
`a block having a series of channels, such as grooves, on a surface
`thereof is utilized.
`The block is placed in contact with a deriva-
`tized glass or other substrate.
`Ina first step, a pipettor or other
`delivery system is used to flow selected reagents to one or more of a
`series of apertures connected to the channels, or place reagents in
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`WO 93/09668
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`the channels directly, filling the channels and “striping” the
`
`substrate with a first reagent, coupling a first group of monomers
`
`thereto.
`
`The first group of monomers need not be homogenous.
`
`For
`
`example, a monomer A may be placed in a first group of the channels,
`
`a monomer B in a second group of channels, and a monomer C in a third
`
`group of channels.
`
`The channels may in some embodiments thereafter
`
`be provided with additional reagents, providing coupling of
`additional monomers to the first group of monomers.
`The block
`is then translated or rotated, again placed on the substrate, and the
`process is repeated with a second reagent, coupling a second group of
`
`monomers to different regions of the substrate.
`
`The process is
`
`repeated until a diverse set of polymers of desired sequence and
`
`By virtue of the process, a
`length is formed on the substrate.
`number of polymers having diverse monomer sequences such as peptides
`or oligonucleotides are formed on the substrate at known locations.
`According to the second aspect of the invention, a series
`of microchannels or microgrooves are formed on a substrate, along
`with an appropriate array of microvalves.
`The channels and valves
`
`The
`are used to flow selected reagents over a derivatized surface.
`microvalves are used to determine which of the channels are opened
`for any particular coupling step.
`Accordingly, one embodiment of the invention provides a
`method of forming diverse polymer sequences on a single substrate,
`the substrate comprising a surface with a plurality of selected
`regions.
`The method includes the steps of forming a plurality of
`channels adjacent the surface,
`the channels at least partially having
`awall thereof defined by a portion of the selected regions; and
`piacing selected reagents in the channels to synthesize polymer
`sequences at the portion of the selected regions,
`the portion of the
`selected regions comprising polymers with a sequence of monomers
`different from polymers in at least one other of the selected
`regions.
`In alternative embodiments,
`the channels or flow paths
`themselves constitute the selected reaction regions.
`For example,
`the substrate may be a series of adjoining parallel channels, each
`having reaction sites therein.
`
`According to a third aspect of the invention, a substrate
`is provided which has an array of discrete reaction regions separated
`from one another by inert regions.
`In one embodiment, a first
`monomer solution is spotted on a first set of reaction regions of a
`suitably derivatized substrate. Thereafter, a second monomer
`
`solution is spotted on a second set of regions, a third monomer
`solution is spotted on a third set and so on, until a number of the
`regions each have one species of monomer located therein. These
`monomers are reacted with the surface, and the substrate is
`
`subsequently washed and prepared for reaction with a new set of
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`WO93/09668
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`PCT/US92/10183
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`4
`
`monomers. Dimers, trimers, and larger polymers of controlled length
`and monomer sequence are prepared by repeating the above steps with
`different groupings of the reaction regions and monomer solutions.
`In alternative embodiments,
`the polymers or other compounds of the
`array are delivered to the regions as complete species, and thus the
`above polymer synthesis steps are unnecessary.
`In a preferred embodiment, a plurality of reaction
`vegions on the substrate surface are surrounded by a constraining
`region such as a non-wetting region which hinders the transport of
`reactants between adjacent reaction regions. Thus, the reactants in
`one region cannot flow to other regions where they could contaminate
`the reaction.
`In certain preferred embodiments, the regions of the
`array are defined by selective irradiation of a substrate surface
`containing photolabile hydrophobic protecting groups.
`In areas where
`the surface is irradiated, the hydrophobic protecting groups are
`removed to define reaction regions. When an aqueous or other polar
`reactant solution is deposited in the reaction region, it will have a
`relatively large wetting angle with the substrate surface so that by
`adjusting the amount deposited, one can ensure no flow to adjacent
`regions.
`
`A further understanding of the nature and advantages of
`the inventions herein may be realized by reference to the remaining
`portions of the specification and the attached drawings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Fig. 1 is a generalized diagram illustrating the
`
`invention;
`
`Fig. 2 igs a flow chart illustrating the treatment steps
`performed in synthesizing an array of various polymers;
`Fig. 3 is a mapping of a resulting array of polymers;
`Fig. 4a to 4¢ illustrate the arrangement of three channel
`block templates in six process steps employed to synthesize 64
`million hexapeptides from a 20 amino acid basis set;
`Fig. 5a is a top view and Fig. 5b is a cross-sectional
`view of a first embodiment of a device used to synthesize arrays of
`
`polymer sequences;
`Fig. 6 is a cross-sectional view of an embodiment
`containing a pressure chamber for holding a substrate against a
`channel block;
`Figs. 7a and 7b are top views of two of two different
`"fanned array" channel blocks;
`Fig. 8 is a cross-sectional view of a channel block and
`associated flow ports according to one embodiment of the invention;
`Fig. 9 is a detailed cross-sectional view of the flow
`ports in a channel block;
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`WO 93/09668
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`5
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`Fig. 10 is a diagram of a flow system used to deliver
`coupling compounds and reagents to a flow cell;
`Figs. lia and 11b show an apparatus used to transfer a
`substrate from one channel block to another;
`Fig. 12 is a diagram of a multichannel solid-phase
`
`synthesizer;
`
`Figs. 13a and 13b illustrate alternative arrangements of
`the grooves in a channel block;
`Fig. 14 is a schematic illustration of reaction pathways
`used to prepare some hydrophobic groups of the present invention;
`Figs. 15a and 15b illustrate a microvalve device;
`Figs. 16a and 16b illustrate an alternative embodiment of
`the invention;
`
`Fig. 17 is a mapping of expected fluorescent intensities
`with a substrate selectively exposed to fluorescent dye.
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`WO 93/09668
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`6
`
`DESCRIPTION OF THE PREFERRED EMBODIMENTS
`
`CONTENTS
`
`I. Glossary
`II. General
`III. Methods for Mechanical
`Delivery of Reagents
`Flow Channel Embodiments
`
`IV.
`
`Vv. Spotting Embodiments
`VI. Alternative Embodiments
`
`VII. Examples
`
`A. Leak Testing
`B. Formation of YGGFL
`
`Cc.
`
`100 Micron Channel Block
`
`D. Channel Matrix Hybridization Assay
`
`VIII. Conclusion
`
`"
`
`e
`
`I.
`
`Glossary
`The following terms are intended to have the following
`general meanings as they are used herein:
`
`i.
`
`2.
`
`A ligand is a molecule that is recognized by a
`Ligand:
`receptor. Examples of ligands that can be investigated by this
`invention include, but are not restricted to, agonists and
`antagonists for cell membrane receptors, toxins and venoms,
`viral epitopes, hormones, opiates, steroids, peptides, enzyme
`substrates, cofactors, drugs, lectins, sugars,
`oligonucleotides, nucleic acids, oligosaccharides, and
`proteins.
`
`
`A monomer is a member of the set of small molecules
`Monomer:
`which are or can be joined together to form a polymer or a
`compound composed of two or more members.
`The set of monomers
`includes but is not restricted to, for example,
`the set of
`common L=amino acids, the set of D-amino acids, the set of
`synthetic and/or natural amino acids,
`the set of nucleotides
`and the set of pentoses and hexoses.
`The particular ordering
`of monomers within a polymer is referred to herein as the
`"sequence" of the polymer. As used herein, monomers refers to
`any member of a basis set for synthesis of a polymer.
`For
`example, dimers of the 20 naturally occurring L-amino acids
`form a basis set of 400 monomers for synthesis of polypeptides.
`Different basis sets of monomers may be used at successive
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`
`steps in the synthesis of a polymer. Furthermore, each of
`the sets may include protected members which are modified after
`synthesis. The invention is described herein primarily with
`regard to the preparation of molecules containing sequences of
`monomers such as amino acids, but could readily be applied
`in the preparation of other polymers.
`Such polymers include,
`for example, both linear and cyclic polymers of nucleic acids,
`polysaccharides, phospholipids, and peptides having either a-,
`B-, or w-amino acids, heteropolymers in which a known drug is
`covalently bound to any of the above, polynucleotides,
`polyurethanes, polyesters, polycarbonates, polyureas,
`polyamides, polyethyleneimines, polyarylene sulfides,
`polysiloxanes, polyimides, polyacetates, or other polymers
`which will be apparent upon review of this disclosure.
`Such
`polymers are "diverse" when polymers having different monomer
`sequences are formed at different predefined regions of a
`substrate. Methods of cyclization and polymer reversal of
`polymers are disclosed in copending application Serial No.
`796,727, filed November 22, 1991, entitled "POLYMER REVERSAL ON
`SOLID SURFACES," incorporated herein by reference for all
`purposes.
`
`3.
`
`4.
`
`A peptide is a polymer in which the monomers are
`Peptide:
`alpha amino acids and are joined together through amide bonds,
`alternatively referred to as a polypeptide. Amino acids may be
`the L-optical isomer or the D-optical
`isomer. Peptides are two
`or more amino acid monomers long and are often more than 20
`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 for all purposes.
`
`A receptor is a molecuie that has an affinity for a
`Receptor:
`ligand. Receptors may be naturally-occurring or manmade
`molecules.
`They can be employed in their unaltered state or as
`- aggregates with other species. Receptors may be attached,
`covalently or noncovalently,
`to a binding member, either
`directly or via a specific 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 antisera reactive with specific
`antigenic determinants, viruses, cells, drugs, polynucleotides,
`nucleic acids, peptides, cofactors,
`lectins, sugars,
`polysaccharides, cellular membranes, and organelles. Receptors
`are sometimes referred to in the art as anti-ligands. As the
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`
`term receptors is used herein, no difference in meaning is
`intended.
`A "Ligand Receptor Pair" is formed when two
`molecules have combined through molecular recognition to form a
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`b)
`
`ce)
`
`complex.
`Specific 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 microorganism receptors such as specific
`transport proteins or enzymes essential to survival of
`microorganisms would be a useful tool for discovering new
`classes of antibiotics. Of particular value would be
`antibiotics against opportunistic fungi, protozoa, and
`bacteria resistant to antibiotics in current use.
`Enzymes: For instance, a receptor can comprise a binding
`site of an enzyme such as an enzyme responsible for
`cleaving a neurotransmitter; determination ef ligands for
`this type of receptor to modulate the action of an enzyme
`that cleaves a neurotransmitter is useful in developing
`drugs that can be used in the treatment of disorders of
`neurotransmission.
`Antibodies:
`For instance, the invention may be useful in
`investigating a receptor that comprises a ligand-binding
`site on an antibody molecule which combines with an
`epitope of an antigen of interest; determining a sequence
`that mimics an antigenic epitope may lead to the
`development of vaccines in 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 therapeutic treatments such as for autoimmune
`diseases (e.g., by blocking the binding of the "self"
`antibodies).
`Nucleic Acids: Sequences of nucleic acids may be
`synthesized to establish DNA or RNA binding sequences
`that act as receptors for synthesized sequence.
`" Catalytic Polypeptides: Polymers, preferably antibodies,
`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 specific 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 and others are described in,-
`for example, PCT Publication No. WO 90/05746,
`
`a)
`
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`9
`
`wo 90/05749, and WO 90/05785, which are incorporated
`herein by reference for all purposes.
`
`f)
`
`Hormone receptors: Determination of the ligands which
`
`bind with high affinity to a receptor such as the
`
`receptors for insulin and growth hormone 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 or a replacement for growth hormone.
`Other examples of hormone receptors include the
`
`vasocconstrictive hormone receptors; determination of
`
`ligands for these receptors may lead to the development
`ef drugs to control blood pressure.
`
`g)
`
`Opiate receptors: Determination of ligands which bind to
`the opiate receptors in the brain is useful in the
`development of less-addictive replacements for morphine
`and related drugs.
`
`A material having a rigid or semi-rigid surface.
`Substrate:
`In many embodiments, at least one surface of the substrate will
`
`be substantially flat, although in some embodiments it may be
`desirable to physically separate synthesis regions for
`different polymers with, for example, wells, raised regions,
`etched trenches, or the like.
`In some embodiments, the
`substrate itself contains wells,
`trenches,
`flow through
`regions, etc. which form all or part of the synthesis regions.
`According to other embodiments, small beads may be provided on
`
`the surface, and compounds synthesized thereon may be released
`upon completion of the synthesis.
`
`A material having a plurality of grooves or
`Channel Block:
`recessed regions on a surface thereof.
`The grooves or recessed
`regions may take on a variety of geometric configurations,
`including but not limited to stripes, circles, serpentine
`paths, or the like. Channel blocks may be prepared in a
`variety of manners,
`including etching silicon blocks, molding
`or pressing polymers, etc.
`
`7.
`
`Protecting Group:
`
`A material which is bound to a monomer unit
`
`and which may be selectively removed therefrom to expose an
`active site such as,
`in the specific example of an amino acid,
`an amine group. Specific examples of photolabile protecting
`groups are discussed in Fodor et al., PCT Publication No. WO
`92/10092 (previously incorporated by reference) and U.S. Serial
`No.
`filed November 2, 1992 (attorney docket No.
`11509-68)
`incerporated herein by reference for all purposes.
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`93.
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`
`A predefined region is a localized area on
`Predefined Region:
`a substrate which is, was, or is intended to be used for
`formation of a selected polymer and is otherwise referred to
`herein in the alternative as "reaction" region, a "selected"
`region, or simply a "region." The predefined region may have
`any convenient shape, e.g., circular, rectangular, elliptical,
`wedge-shaped, etc.
`In some embodiments, a predefined region
`and, therefore, the area upon which each distinct polymer
`sequence is synthesized is smailer than about 1 cm’, more
`preferably less than 1 mm, and still more preferably less than
`0.5 mm.
`In most preferred embodiments the regions have an
`area less than about 10,000 um* or, more preferably,
`less than
`100 uwm*. Within these regions,
`the polymer synthesized therein
`is preferably synthesized in a substantially pure form.
`
`A polymer is considered to be
`Substantially Pure:
`"substantially pure" within a predefined region of a substrate
`when it exhibits characteristics that distinguish it from other
`predefined regions. Typically, purity will be measured in
`terms of biological activity or function as a result of uniform
`sequence.
`Such characteristics will typically be measured by
`way of binding with a selected ligand or receptor. Preferably
`the region is sufficiently pure such that the predominant
`species in the predefined region is the desired sequence.
`According to preferred aspects of the invention,
`the polymer is
`at least 5% pure, more preferably more than 10% to 20% pure,
`more preferably more than 80% to 90% pure, and most preferably
`more than 95% pure, where purity for this purpose refers to the
`ratio of the number of ligand molecules formed in a predefined
`region having a desired sequence to the total number of
`molecules foxmed in the predefined region.
`
`II. General
`The invention can be used in variety of applications.
`the invention can be used as a synthesis tool (as for
`For example,
`example in peptide syntheses), as a screening tool (as for example in
`screening compound libraries for drug activity), or as a
`monitoring/diagnostic tool
`(as for example in medical or
`environmental testing).
`In one specific embodiment,
`the invention is
`used for nucleic acid-based diagnostics.
`As a synthesis tool,
`the present invention provides for
`the formation of arrays of large numbers of different polymer
`sequences. According to a preferred embodiment,
`the invention
`provides for the synthesis of an array of different peptides or
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`Such substrates
`Oligonucleotides in selected regions of a substrate.
`having the diverse sequences formed thereon may be used in, for
`example, screening studies to evaluate their interaction with
`
`in
`
`For example,
`receptors such as antibodies and nucleic acids.
`preferred embodiments the invention provides for screening of
`peptides to determine which if any of a diverse set of peptides has a
`strong binding affinity with a receptor and,
`in most preferred
`embodiments,
`to determine the relative binding affinity of various
`peptides with a receptor of interest.
`Such diverse polymer sequences are preferably synthesized
`on a single substrate.
`By synthesizing the diverse polymer sequences
`on a single substrate, processing of the sequences to evaluate
`characteristics such as relative binding affinity is more easily
`conducted.
`By way of example, when an array of peptide sequences (or
`a library of other compounds)
`is to be evaluated to determine the
`peptides’ relative binding affinity to a receptor,
`the entire
`substrate and,
`therefore, all or a group of the polymer sequences may
`be exposed to an appropriately labelled receptor and evaluated
`simultaneously.
`
`the present invention can be
`In some embodiments,
`employed to localize and,
`in some cases,
`immobilize vast collections
`of synthetic chemical compounds or natural product extracts.
`In such
`methods, compounds are deposited on predefined regions of a
`substrate.
`The reaction of the immobilized compound (or compounds)
`with various test compositions such as the members of the chemical
`library or a biological extract are tested by dispensing small
`aliquots of each member of the library or extract to a different
`region. Competitive assays or other well-known techniques can be
`used to identify a desired activity. As an example, a large
`collection of human receptors is deposited on a substrate, one in
`each region to form an array.
`A plant/animal extract is then
`screened for binding to various receptors of the array.
`The present invention has certain features in common with
`the “light directed" methods described in U.S. Patent No. 5,143,854,
`previously incorporated by reference.
`The light directed methods
`discussed in the ‘854 patent involve activating predefined regions of
`the substrate and then contacting the substrate with a preselected
`monomer solution.
`The predefined regions can be activated with a
`light source shown through a mask (much in the manner of
`photolithography techniques used in integrated circuit fabrication).
`Other regions of the substrate remain inactive because they are
`blocked by the mask from illumination. Thus, a light pattern defines
`which regions of the substrate react with a given monomer.
`By
`repeatedly activating different sets of predefined regions and
`contacting different monomer solutions with the substrate, a diverse
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`array of polymers is produced on the substrate. Of caurse, other
`steps such as washing unreacted monomer solution from the substrate
`can be used as necessary.
`In the present invention, a mechanical device or physical
`structure defines the regions which are available to react with a
`given monomer.
`In some embodiments, a wall or other physical barrier
`is used to block a given monomer solution from contacting any but a
`few selected regions of a substrate.
`In other embodiments, the
`amount of the monomer (or other) solution deposited and the
`composition of the substrate act to separate different monomer
`solutions on the substrate. This permits different monomers to be
`delivered and coupled to different regions simultaneously (or nearly
`simultaneously) and reduces the number of separate washing and other
`reaction steps necessary to form an array of polymers. Further, the
`reaction conditions at different activated regions can be controlled
`independently. Thus,
`the reactant concentrations and other
`parameters can be varied independently from- reaction site to reaction
`site, to optimize the procedure.
`In alternative preferred embodiments of the present
`invention, light or another activator is used in conjunction with the
`physical structures to define reaction regions.
`For example, a light
`gource activates various regions of the substrate at one time and
`then a mechanical system directs monomer solutions to different
`activated regions,
`in parallel.
`
`Iiz.
`
`Methods for Mechanical Delivery of Reagents
`In preferred embodiments of the present invention,
`reagents are delivered to the substrate by either (1) flowing within
`a channel defined on predefined regions or (2) “spotting” on
`predefined regions. However, other approaches, as well as
`combinations of spotting and flowing, may be employed.
`In each
`instance, certain activated regions of the substrate are mechanically
`separated from other regions when the monomer solutions are delivered
`to the various reaction sites.
`A typical "flow channel" method of the present invention
`can generally be described as follows. Diverse polymer sequences are
`synthesized at selected regions of a substrate by forming flow
`channels on a surface of the substrate through which appropriate
`reagents flow or in which appropriate reagents are placed. For
`example, assume a monomer "A" is to he bound to the substrate ina
`first group of selected regions.
`If necessary, all or part of the
`surface of the substrate in all or a part of the selected regions is
`activated for binding by, for example,
`flowing appropriate reagents
`through all or some of the channels, or by washing the entire
`substrate with appropriate reagents. After placement of a channel
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`block on the surface of the substrate, a reagent having the monomer A
`
`The
`flows through or is placed in all or some of the channel(s).
`thereby
`channels provide fluid contact to the first selected regions,
`binding the monomer A on the substrate directly or indirectly (via a
`linker) in the first selected regions.
`
`Thereafter, a monomer B is coupled to second selected
`
`some of which may be included among the first selected
`regions,
`The second selected regions will be in fluid contact with a
`regions.
`second flow channel(s) through translation, rotation, or replacement
`
`of the channel biock on the su