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WO2011071943A1 - Matrices de présentation de peptide - Google Patents

Matrices de présentation de peptide Download PDF

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Publication number
WO2011071943A1
WO2011071943A1 PCT/US2010/059327 US2010059327W WO2011071943A1 WO 2011071943 A1 WO2011071943 A1 WO 2011071943A1 US 2010059327 W US2010059327 W US 2010059327W WO 2011071943 A1 WO2011071943 A1 WO 2011071943A1
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WO
WIPO (PCT)
Prior art keywords
peptide
oligonucleotide
array
region
interest
Prior art date
Application number
PCT/US2010/059327
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English (en)
Inventor
Mark S. Chee
Igor A. Kozlov
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Prognosys Biosciences, Inc.
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Publication date
Application filed by Prognosys Biosciences, Inc. filed Critical Prognosys Biosciences, Inc.
Priority to US13/514,045 priority Critical patent/US20120270748A1/en
Priority to AU2010328226A priority patent/AU2010328226A1/en
Priority to JP2012543208A priority patent/JP2013512691A/ja
Priority to EP10836568.5A priority patent/EP2510127B1/fr
Priority to CN2010800553512A priority patent/CN102648294A/zh
Publication of WO2011071943A1 publication Critical patent/WO2011071943A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1075—Isolating an individual clone by screening libraries by coupling phenotype to genotype, not provided for in other groups of this subclass
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B80/00—Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00533—Sheets essentially rectangular
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/0074—Biological products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag

Definitions

  • This invention relates to peptide-based arrays, methods of producing such arrays, and related methods of use.
  • Protein microarrays are a very useful tool for such high throughput analysis of proteins, but the availability of microarray technology for large scale proteomics studies is still very limited due to the difficulty and cost of protein production (Henderson G and Bradley M, Curr Opin Biotechnol. Aug;18(4):326-30, (2007), Epub 2007 Aug 6; Tapia VE Methods Mol Biol. 2009;570:3-17 (2009)).
  • peptide arrays are made by spotting pre-synthesized peptides on a surface (Salisbury CM et al, J Am Chem Soc. 2002 Dec 18; 124(50): 14868-70 (2002)) or by synthesizing peptides in spots on cellulose filter sheets using standard solid phase peptide synthesis, also known as the SPOT method (Frank R, J Immunol Methods. 2002 Sep 1;267(1): 13-26 (2002)).
  • the cost of generating arrays with tens to hundreds of thousands of peptides is very high, making large-scale, high throughput uses of such arrays cost prohibitive.
  • the present invention provides arrays, methods of constructing arrays, and methods of use of such arrays.
  • the arrays of the invention comprise a substrate with two or more discrete constructs or discrete sets of constructs associated on the surface of the substrate, optionally via a linker molecule.
  • the constructs comprise both an oligonucleotide encoding a peptide of interest and the peptide of interest itself.
  • the oligonucleotide is associated with the substrate surface, and the peptide is associated with the oligonucleotide, either directly or via a linker molecule.
  • the constructs of the array include a first oligonucleotide region encoding a peptide of interest and an affinity tag, a second oligonucleotide region positioned 5' to the first oligonucleotide comprising an untranslated region, a fusion peptide comprising both the peptide of interest and the affinity tag; and a capture agent associated with the oligonucleotide that forms a binding pair with the affinity tag of the fusion protein.
  • the untranslated region of the oligonucleotide comprises a transcriptional start site and a ribosomal binding site 5 ' to the region encoding the peptide of interest and the affinity tag.
  • the capture agent may be associated with the oligonucleotide directly or via a linker molecule.
  • the fusion peptide is associated with the oligonucleotide at the terminus of the oligonucleotide distal to the substrate surface via binding to a capture agent associated directly or indirectly with the oligonucleotide.
  • the peptide is associated with the oligonucleotide near the surface of the substrate, either via a capture agent associated with the oligonucleotide or a capture agent associated with the linker to which the oligonucleotide attaches to the substrate surface.
  • the capture agent is associated at a position on the oligonucleotide distal to the substrate surface via a primer sequence linked to the oligonucleotide, e.g., using a synthetic linker molecule.
  • the array substrate is a planar support, a film, beads or a combination thereof.
  • One strand of the oligonucleotide comprises a primer region used for introduction of an untranslated region to the oligonucleotide.
  • the untranslated region comprises a ribosomal start site and a transcriptional promoter region for the initiation of the transcription reaction.
  • the constructs are arrayed on the surface of a flow cell, and preferably a flow cell used for high throughput sequencing.
  • the sequences can optionally be clonally amplified in situ in the flow cell.
  • the sequences are arrayed randomly, but the identity of each clonal sequence can be determined either before or after the production of protein and its use in a screening assay.
  • the ability to array many millions of DNA templates and determine their sequence can be done quite inexpensively, e.g., by randomization of bases at defined positions during synthesis of an oligo template, by combining shorter oligos to form a longer template, or by deriving a library of sequences from a genomic DNA or cDNA library.
  • the constructs of the arrays comprise double-stranded oligonucleotides associated with the peptide. In other aspects, it may be desirable to remove one of the oligonucleotide strands following peptide association, and the arrays will comprise a single-stranded oligonucleotide associated with the peptide of interest.
  • the invention also provides methods of constructing an array of the invention.
  • Specific methods of producing peptide arrays of the invention utilize sets of nucleic acids synthesized on a substrate surface (e.g., a planar substrate or a set of beads).
  • Nucleic acids of known sequence encoding amino acid sequences are preferably synthesized directly onto the surfaces, e.g., using chemical synthesis techniques.
  • the identity of the sequences at each different location or site on the array may be predetermined or determined following synthesis on the solid substrate or assembly (e.g., in the case of beads) into a fixed format, such as a microarray.
  • the sequence identities may also be determined after construction of the peptide array.
  • nucleic acids comprising desired sequence information (e.g., sequences encoding elements of the peptide array, sequences necessary for transcription or translation, and the like) are attached to the synthesized nucleic acids on the substrate surface using methods such as chemical and preferably enzymatic ligation, primer extension, amplification, etc.
  • the extended nucleic acids are used as templates for the production of peptides of interest and other peptide elements (e.g., affinity tags) via in vitro transcription and translation.
  • nucleic acid microarrays comprising a set of chemically synthesized oligonucleotides encoding amino acid sequences are manufactured. Additional nucleic acids comprising desired sequence information (e.g., sequences encoding elements of the peptide array, sequences necessary for transcription or translation, and the like) are attached to the oligonucleotides of these arrays using methods such as chemical and preferably enzymatic ligation, primer extension, amplification, etc. The extended nucleic acids are used as templates for the production of peptides of interest and other peptide elements (e.g., affinity tags) via in vitro transcription and translation.
  • desired sequence information e.g., sequences encoding elements of the peptide array, sequences necessary for transcription or translation, and the like
  • the extended nucleic acids are used as templates for the production of peptides of interest and other peptide elements (e.g., affinity tags) via in vitro transcription and translation.
  • the peptides are captured at known locations on the substrate surface.
  • the peptides are associated with the locations of their respective nucleic acid templates, or directly with the actual templates, using the methods described herein in more detail.
  • These methods of the invention utilizing a combination of synthetic and post-synthetic nucleic acid construction are particularly useful in the creation of large numbers of peptides of known sequence on a substrate surface. For example, using these methods, arrays of at least 100, at least 1000, at least 5000 and at least 10,000 resolvable peptides can be produced on a substrate surface.
  • the extended nucleic acid sequences are transferred (either directly or as copies) to the surface and amplified in situ, where the rest of the process of peptide array construction is carried out.
  • the arrays can be produced using a surface comprising two or more constructs comprising a single-stranded oligonucleotide template region encoding a peptide of interest and an affinity tag.
  • single- stranded oligonucleotide templates are converted into a double-stranded oligonucleotide region, and an untranslated region comprising a transcription start site and a ribosomal binding site is introduced to the 5 ' end of the double-stranded oligonucleotide region.
  • the untranslated region is added to the single-stranded region prior to conversion of the single-stranded oligonucleotide to a double-stranded template.
  • the double- stranded oligonucleotide region is then subjected to an in vitro transcription and translation event to produce a fusion peptide comprising the peptide of interest and the affinity tag, and the fusion peptide is captured in the construct by binding of the affinity tag portion of the fusion peptide to a capture agent associated directly or indirectly with the oligonucleotide.
  • the capture agent is optionally associated with the untranslated region of the oligonucleotide and is introduced to the oligonucleotide via a region complementary to a region of this untranslated area.
  • the capture agent may be associated with the construct via the peptide coding region or via a linker attaching the oligonucleotide template to the substrate surface.
  • the arrays can be produced using a surface comprising two or more constructs comprising a single-stranded oligonucleotide template region comprising a sequence complementary to a sequence encoding a peptide of interest and complementary to a sequence encoding an affinity tag.
  • the single- stranded oligonucleotide templates are converted into a double-stranded oligonucleotide region, and an untranslated region comprising a transcription start site and a ribosomal binding site is introduced to the 5 ' end of the double-stranded oligonucleotide region.
  • the double-stranded oligonucleotide region is then subjected to a transcription and translation event to produce a peptide comprising the peptide of interest and the affinity tag, and the fusion peptide is captured in the construct by binding of the affinity tag of the fusion peptide to a capture agent.
  • the capture agent optionally is associated with the untranslated region and introduced to the construct with this region. Alternatively, the capture agent may be associated with the construct via the peptide encoding region or via a linker attaching the oligonucleotide to the substrate surface.
  • the invention provides a method of constructing an array by providing a substrate comprising a surface with two or more constructs comprising a single-stranded oligonucleotide encoding a peptide of interest and an affinity tag.
  • a second, universal oligonucleotide comprising an untranslated region with a transcriptional start site and a ribosomal binding site is attached to the oligonucleotides of the construct, and a capture agent is associated with the oligonucleotides via hybridization to the universal oligonucleotide.
  • the single- stranded oligonucleotide regions of the constructs are converted into double- stranded oligonucleotide regions; and the double-stranded oligonucleotide template region is subjected to a transcription and translation event to produce a fusion peptide comprising the peptide of interest and the affinity tag.
  • the fusion protein produced from this translation event is captured by the capture agent via the peptide's affinity tag.
  • the resulting array comprises discrete units comprising a nucleic acid associated with the peptide encoded by the nucleic acid. These discrete construct units can comprise one or more constructs that are distinguishable from other constructs having separate peptides of interest.
  • the invention provides a method of constructing an array by providing a substrate comprising a surface with two or more constructs comprising a single-stranded oligonucleotide encoding a peptide of interest and an affinity tag.
  • a second, universal oligonucleotide comprising an untranslated region with a transcriptional start site and a ribosomal binding site is attached to the oligonucleotides of the construct, and a capture agent is associated with the oligonucleotides via hybridization to the universal oligonucleotide.
  • the single- stranded oligonucleotide regions of the constructs are converted into double- stranded oligonucleotide regions; and the double-stranded oligonucleotide template region is subjected to a transcription and translation event to produce a fusion peptide comprising the peptide of interest and the affinity tag.
  • the fusion protein produced from this translation event is captured by the capture agent via the peptide's affinity tag.
  • the resulting array comprises discrete units comprising a nucleic acid associated with the peptide encoded by the nucleic acid. These discrete construct units can comprise one or more constructs that are distinguishable from other constructs having separate peptides of interest.
  • the invention provides a method of constructing an array by providing a substrate comprising a surface with two or more constructs comprising a single-stranded oligonucleotide comprising a region encoding a peptide of interest and an affinity tag and an untranslated region, where the oligonucleotide is attached to the substrate surface at its 5' end.
  • Primer annealing to the untranslated region of the oligonucleotide creates a double stranded region at the transcriptional starts site, which is efficient to initiate transcription and translation of the peptide encoding the peptide of interest and the affinity tag.
  • the constructs of the array are not converted into a double-stranded oligonucleotide prior to the initiation of transcription and translation.
  • the invention also provides methods for detecting binding of an agent to a peptide of interest, including detection of the presence or absence of an agent in a sample. These methods comprise providing an array comprising: a substrate having a surface; two or more individual constructs associated on the surface of the substrate, the individual constructs having both an oligonucleotide encoding a peptide of interest and the peptide of interest itself.
  • the arrays used in these detection methods have two or more constructs comprising: an oligonucleotide region encoding a peptide of interest, an oligonucleotide region encoding an affinity tag, and an untranslated region; a peptide comprising the peptide of interest and an affinity tag; and a capture agent that selectively binds to the affinity tag.
  • Detection of the binding of an agent to the peptide of interest is accomplished by exposing the array to an agent and detecting the presence or absence of binding of the agent to the peptide of interest.
  • the individual constructs are associated with the array through the association of the oligonucleotide with the substrate surface, and the peptide is associated with the oligonucleotide e.g., through binding of the affinity tag to the capture agent.
  • FIGs. 1A-1F illustrates exemplary constructs that are used in the composite oligonucleotide-peptide arrays of the invention.
  • FIG. 2 illustrates an array comprising sets of identical constructs comprising the same fusion peptides.
  • FIG. 3 illustrates an array comprising sets of constructs comprising the same oligonucleotide region in different configurations.
  • FIG. 4 illustrates an array comprising constructs with the same peptide but with different affinity tags and capture agents.
  • FIG. 5 illustrates a first general approach for producing the composite oligonucleotide-peptide arrays of the invention.
  • FIG. 6 illustrates a second general approach for producing the composite oligonucleotide-peptide arrays of the invention using capture agents.
  • FIG. 7 illustrates a first production method using capture agents and oligonucleotide templates on beads.
  • FIG. 8 illustrates a second production method using capture agents and oligonucleotide templates on a planar surface.
  • FIG. 9 illustrates a third production method using puromycin capture of the peptide produced by the oligonucleotide template.
  • FIG. 10 illustrates a more specific aspect of the production method of FIG.
  • FIG. 11 illustrates the LCTPSR peptide using FGE using the method illustrated in FIG. 10.
  • FIG. 12 illustrates a general assay system utilizing a sequencing flow cell.
  • FIG. 13 illustrates the use of various construct configurations on an array of the invention.
  • FIG. 14 illustrates the use of diffusion to create a halo effect to reduce false positives on an array of the invention.
  • FIG. 15 illustrates the peptide binding results obtained using the bead arrays of FIG. 7.
  • FIG. 16 illustrates the peptide binding results obtained using the slide arrays produced as shown in FIG. 8.
  • affinity tag refers to one member of a binding pair that selectively binds to a capture agent.
  • binding pair means any two molecules that are known to selectively bind to one another. In the case of two proteins, the molecules selectively bind to one another as described in more detail herein. Such binding may include covalent and/or non-covalent binding. Examples include, but are not limited to, biotin and avidin; biotin and streptavidin; an antibody and its particular epitope; and the like.
  • capture agent refers any moiety that allows capture of a peptide via binding to or linkage with an affinity tag of the peptide.
  • the binding between the capture agent and its affinity tag may be a covalent bond and/or a non-covalent bond.
  • a capture agent includes, e.g., a member of a binding pair that selectively binds to an affinity tag on a fusion peptide, a chemical linkage that is added by recombinant technology or other mechanisms, and the like.
  • the capture agent is an antibody that selectively binds to an affinity tag epitope.
  • Capture agents can be associated with a construct using conventional techniques including hybridization, crosslinking (e.g., covalent immobilization using psoralen), introduction through post- translational modification and the like.
  • complementary refers to the topological compatibility or interactive structure of interacting surfaces of a nucleic acid binding pair.
  • Preferred complementary structures have binding affinity for each other and the greater the degree of complementarity the nucleic acids have for each other the greater the hybridization between the structures.
  • diagnostic tool refers to any composition or assay of the invention used in order to carry out a diagnostic test or assay on a patient sample.
  • the composition of the invention may be considered a collection of analyte specific reagents, and as such may form part of a diagnostic test regulated by a federal or state agency.
  • the use of the compositions of the invention as a diagnostic tool is not intended to be related to any use of the composition in the development of therapeutic agents.
  • oligonucleotide is used herein to mean a linear polymer of nucleotide monomers. As used herein, the term may refer to single stranded or double stranded forms. Monomers making up nucleic acids and oligonucleotides are capable of specifically binding to a natural polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like, to form duplex or triplex forms.
  • Such monomers and their internucleosidic linkages may be naturally occurring or may be analogs thereof, e.g., naturally occurring or non-naturally occurring analogs.
  • Non-naturally occurring analogs may include peptide nucleic acids, locked nucleic acids, phosphorothioate internucleosidic linkages, bases containing linking groups permitting the attachment of labels, such as fluorophores, or haptens, and the like.
  • oligonucleotide or nucleic acid requires enzymatic processing, such as extension by a polymerase, ligation by a ligase, or the like, one of ordinary skill would understand that oligonucleotides or nucleic acids in those instances would not contain certain analogs of internucleosidic linkages, sugar moieties, or bases at any or some positions, when such analogs are incompatible with enzymatic reactions.
  • Nucleic acids typically range in size from a few monomeric units, e.g., 5-40, when they are usually referred to as "oligonucleotides," to several hundred thousand or more monomeric units.
  • nucleic acid or oligonucleotide is represented by a sequence of letters (upper or lower case), such as "ATGCCTG,” it will be understood that the nucleotides are in 5'>3' order from left to right and that "A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes deoxythymidine, “I” denotes deoxyinosine, "U” denotes uridine, unless otherwise indicated or obvious from context.
  • A denotes deoxyadenosine
  • C denotes deoxycytidine
  • G denotes deoxyguanosine
  • T denotes deoxythymidine
  • I denotes deoxyinosine
  • U denotes uridine, unless otherwise indicated or obvious from context.
  • nucleic acids comprise the natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs, e.g., modified bases, sugars, or internucleosidic linkages.
  • natural nucleosides e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA
  • non-natural nucleotide analogs e.g., modified bases, sugars, or internucleosidic linkages.
  • an enzyme has specific oligonucleotide or nucleic acid substrate requirements for activity, e.g., single-stranded DNA, RNA/DNA duplex, or the like
  • selection of appropriate composition for the oligonucleotide or nucleic acid substrates is well within the knowledge of one of ordinary skill, especially with guidance from treatises, such as Sambrook et al., Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989), and like references.
  • peptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • search tool refers to any composition or assay of the invention used for scientific enquiry, academic or commercial in nature, including the development of pharmaceutical and/or biological therapeutics.
  • the research tools of the invention are not intended to be therapeutic or to be subject to regulatory approval; rather, the research tools of the invention are intended to facilitate research and aid in such development activities, including any activities performed with the intention to produce information to support a regulatory submission.
  • binding partner e.g., protein, nucleic acid, antibody, etc.
  • binding partner refers to a binding reaction of two or more binding partners with high affinity and/or complementarity to ensure selective hybridization under designated assay conditions.
  • specific binding will be at least three times background signal or noise and more typically more than 10 to 100 times background.
  • the binding partner binds to its particular "target” molecule and does not bind in a significant amount to other molecules present in the sample.
  • T m is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • T m is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • the arrays of the present invention provide novel array compositions and methods of producing these arrays in a cost-effective manner.
  • the arrays of the invention comprise both an oligonucleotide peptide-coding template, and preferably a double-stranded oligonucleotide peptide-coding template, associated with a peptide encoded by the oligonucleotide template.
  • the microarrays of the invention are produced using an in vitro transcription/translation of oligonucleotide templates obtained by very inexpensive microarray based synthesis.
  • the oligonucleotides of the array are converted into oligo-peptide composite constructs by transcribing the oligonucleotides attached to the array surface into RNA templates followed by translation of these templates into peptides.
  • the translated peptide is then associated to the oligonucleotide template via different mechanisms, including the use of a chemical linker group, the use of a capture agent, and the like.
  • the variety of peptides available on the array reflects the complexity of the original oligonucleotide templates on the array.
  • the arrays comprise a capture agent that is used to bind to an affinity tag region on the peptide.
  • the peptide is a fusion peptide comprising an affinity tag region which binds the capture agent at the C-terminal region, and the peptide(s) of interest at the N- terminal region.
  • the affinity tag is encoded by the oligonucleotide template in the construct, and is used to capture the fusion peptide created through transcription and translation.
  • multiple peptides on the array will comprise the same affinity tag, allowing the use of a common capture agent to be used in the constructs.
  • an array may comprise peptides of varying sequence having a single common affinity tag and capture agent. This allows the use of a single peptide binding moiety to be added during the construction of the array, significantly decreasing the complexity and cost of production.
  • the array may comprise two or more constructs having the same peptide sequence of interest, but with different affinity tags and capture agents. This latter approach allows formation of arrays having internal control mechanisms to ensure that the activity of the peptide of interest is not adversely affected by the process of constructing or using the array.
  • the methods of protein microarray production of the instant invention are reliable and reproducible, and capable of producing identical arrays of the same quality and protein quantity.
  • the arrays of the invention are useful in the display of a wide variety of peptides, including proteins and fragments thereof from various classes and sizes.
  • the yield of protein per array unit is sufficiently high to allow reproducible and discrete detection and/or activity for the particular array units. This allows the arrays to be produced on a large scale with extremely high density at a relatively low cost.
  • the arrays of the invention have the ability to display functional peptides, including entire proteins, peptide domains, active sites of proteins, and the like. It is a feature of the invention that the peptide arrays can be created to study functional proteins that are generally difficult to isolate from in vivo sources, e.g., insoluble proteins such as prions or beta amyloid peptides.
  • the arrays of the invention comprise double-stranded or single-stranded oligonucleotides associated with both a substrate surface and a peptide of interest.
  • the array construct comprises an oligonucleotide portion 102 comprising an untranslated region 110, a region 112 encoding a peptide of interest, and a region 114 encoding an affinity tag and a stop codon.
  • the oligonucleotide may be directly associated with the surface, or optionally attached to the array using a linker 106.
  • a single strand is attached to the array surface, and the strand may be attached at either its 3 Or 5' end.
  • the portion of the oligonucleotide distal to the array comprises a peptide association moiety 116 which allows the peptide produced from the oligonucleotide template to be associated with the construct following in vitro transcription and translation.
  • the associated peptide 150 comprises the peptide of interest 120 which is encoded by the coding region 112 of the oligonucleotide, fused to the affinity tag 122 which is encoded by region 114 of the oligonucleotide.
  • the affinity tag 122 allows attachment of the peptide 150 to the oligonucleotide.
  • the peptide association moiety 116 comprises a capture agent, e.g., an antibody or a member of a protein binding pair.
  • the oligonucleotide construct may have a strand removed following transcription and translation, resulting in a single- stranded molecule 104 with either a sequence encoding the peptide or a sequence complementary to the sequence encoding the peptide.
  • This single strand may be attached to the substrate surface at either its 3 Or 5' end.
  • FIG. ID illustrates a construct having the same components as those in FIG. IB, but in which the orientation of the oligonucleotide 102 is effectively flipped.
  • the capture agent 116 is associated proximal to the substrate surface relative to the oligonucleotide.
  • the capture agent 116 can be associated with the construct via either the linker 106 that attaches the oligonucleotide portion to the substrate surface 108 (as illustrated in IE) or via the oligonucleotide 102 itself (as illustrated in IF).
  • the oligonucleotide constructs 102 shown here may be either single-stranded as illustrated in FIG. 1C, or double- stranded, as illustrated in FIGs. 1A, IB and ID. Generally, a single strand is attached to the array surface, and the strand may be attached to the substrate surface at either its 3 Or 5' end.
  • the arrays may contain individual constructs that themselves are discrete, interrogatable units for analysis of the peptide of interest. For certain applications, however, it is desirable to increase the intensity of signal created on the array.
  • the invention thus also includes arrays having discrete interrogatable units comprising two or more identical constructs that display the same peptide of interest.
  • the array comprises a substrate having different constructs positioned on the array in discrete groups of two or more constructs, which preferably utilize the same capture agents and affinity tags for association of the peptides to the oligonucleotide.
  • the constructs of the first set comprise an oligonucleotide portion 202 comprising an untranslated region 210, a region 212 encoding a first peptide of interest, and a region 214 encoding an affinity tag and a stop codon.
  • the constructs of the second set comprise an oligonucleotide portion 204 comprising an untranslated region 210, a region 232 encoding a second peptide of interest, and a region 214 encoding an affinity tag and a stop codon.
  • the oligonucleotides of both sets may be directly associated with the surface, or optionally attached to the array using linkers 206.
  • the portions of the oligonucleotide distal to the array comprises peptide association moieties 216 which allows the peptides produced from the oligonucleotide templates to be associated with the construct following in vitro transcription and translation.
  • the associated peptides 250 of the first set comprise the first peptide of interest 220 fused to the affinity tag 222 which is encoded by 214.
  • the associated peptides of the second set 252 comprise the second peptide of interest 240 fused to the same affinity tag 222 as the first set, which as in the first construct is encoded by a common region 214.
  • the array may comprise individual constructs that have the same oligonucleotide regions in a different relationship with respect to the substrate.
  • the constructs have the same components as the constructs of FIGs. IB and ID, with one set of constructs having at least one construct on the array in the configuration of FIG. IB and a second set of constructs having at least one construct on the array in the configuration of FIG. ID.
  • the constructs of the first set comprise an oligonucleotide portion 302 comprising an untranslated region 310, a region 312 encoding a first peptide of interest, and a region 314 encoding an affinity tag and a stop codon, with the latter region 314 being attached to the substrate surface.
  • the constructs of the second set comprise an oligonucleotide portion 304 comprising the untranslated region 310, region 312 encoding a first peptide of interest, and region 314 encoding an affinity tag and a stop codon, but in this configuration the first region 310 is attached to the substrate surface.
  • the oligonucleotides of both sets may be directly associated with the substrate surface, or optionally attached to the array using linkers 306, and they may be attached to the surface via either the 3' or the 5' end.
  • the portions of the oligonucleotides distal to the substrate surface comprise peptide association moieties 316 which allow the peptides produced from the oligonucleotide templates to be associated with the construct following in vitro transcription and translation.
  • the illustrated array comprises individual constructs comprising oligonucleotides that encode the same peptides of interest, but where each peptide is associated with two different capture agents on the oligonucleotide.
  • a first set of oligonucleotides 402 and 452 comprise the same untranslated region 410 and region 412 encoding a first peptide of interest 420, but they are linked to different regions 414 and 444 encoding different affinity tags.
  • peptides produced from these two constructs will comprise the same peptide of interest, but fused to two different affinity tags that are captured using two different capture agents 416, 446.
  • a second set of oligonucleotides 404 and 454 comprise the same untranslated region 410 and region 442 encoding a second peptide of interest 440, but they are linked to different regions 414 and 444 encoding different affinity tags 422, 432.
  • the arrays of the invention can be produced using initial made-to-order oligonucleotide arrays using any of a number of technologies.
  • the arrays can be produced on a planar surface, or on a series of discrete surfaces, e.g. beads, that together form an array.
  • the composite oligo-peptide arrays can be produced using any single-stranded or double-stranded oligonucleotide array.
  • Suitable methods for such oligonucleotide array production can be found, for example, in US Pat. No. 7,556,919, issued July 2009 to Chenchik et al.; US Pat. No. 7,291,471 issued November 2007 to Sampson et al.; US Pat. No. 6,294,336, issued September 2001 to Boyce-Jacino et al.; US Pat. No. 6,291, 183, issued September 2001 to Pirrung et al.; US Pat. No. 6,284,497 issued September 2001 to Sabanayagam et al.; US Pat. No. 6,261,776 issued July 2001 to Pirrung et al.; US Pat. No.
  • oligonucleotide templates can be synthesized in parallel on an array.
  • the single-stranded oligonucleotides comprise universal primer sequences corresponding to an untranslated region or a region complementary to a universal untranslated region.
  • the oligonucleotide is associated with a region for the capture of the peptide following translation.
  • the oligonucleotide can be synthesized to have each of these elements, or an oligonucleotide can be constructed on the substrate by addition of the various elements to an initial oligonucleotide associated with the substrate surface.
  • the oligonucleotides used in construction of the array comprise regions encoding an affinity tag and stop codon at one end of the oligonucleotide and a primer region used to attach a long universal untranslated region at the other end of the oligonucleotide.
  • the single-stranded oligonucleotide also comprises a region complementary to a primer that is used to synthesize a strand complementary to the single-stranded oligonucleotides, resulting in extension of the single-stranded oligonucleotide into a double- stranded oligonucleotide template for use in the in vitro transcription and translation reactions.
  • the oligonucleotides will comprise the primer for attachment of the untranslated region at the 5' end of the oligonucleotide and the sequence encoding the affinity tag at the 3' end of the oligonucleotide.
  • the substrate-bound single-stranded oligonucleotides may comprise the reverse complement sequences, and the strand complementary to the initial oligonucleotide will be transcribed. In the latter case, single-stranded oligonucleotides comprising sequences complementary to the untranslated region and the affinity tag are initially bound to the substrate.
  • peptide fragments or peptides having deletions will be selected against, as fusion peptides expressed from the templates that contain deletions or insertions (e.g., from byproducts of oligonucleotide synthesis or mistakes in transcription) will generally not be captured on the array because they will have a frame shift during translation and will not display the correct sequence of the affinity tag.
  • the untranslated region preferably comprises a transcriptional promoter region at the 5 '-end followed by a ribosomal binding site (RBS) that is used to initiate the transcription and translation events to produce the peptides of the array.
  • the untranslated sequence can be included in the initially synthesized oligonucleotide, or it can be attached to the oligonucleotide template using various techniques.
  • the 5' primer region of the oligonucleotide may comprise a restriction endonuclease site, and the universal untranslated region can be added to the 5' of the oligonucleotide by digestion and ligation.
  • a primer complementary to both the 5' primer region and the universal untranslated region can be used for splint ligation of the two molecules.
  • FIG. 5 illustrates a general scheme for production of the arrays of the invention using single-stranded oligonucleotide arrays.
  • the method begins with oligonucleotides 500 arrayed on a surface 508, optionally attached to the surface via a linker sequence 506.
  • the oligonucleotides 500 each comprise a first region 514 that encodes an affinity tag and a stop codon, and which comprises a sequence that hybridizes selectively to a primer used in primer extension of the oligonucleotide, a coding region 512 encoding a peptide or complementary to a region encoding a peptide, and a second primer region 510 for attachment of a universal untranslated region 518 and/or a capture agent 516 for the encoded peptide.
  • the capture agent 516 may be a chemical linkage group that interacts with and binds to the affinity tag.
  • Extension of the single-stranded molecule from the primer region 514 and attachment of the untranslated region in step 511 produces a double-stranded template 502 that includes the capture agent 516 on the construct positioned distally to the substrate 508.
  • the coding region 512 encodes the peptide of interest
  • the primer extension region 514 comprising a coding region for an affinity tag that binds to a capture agent 516 and a stop codon.
  • In vitro transcription and translation will produce the fusion peptide 550, comprising both the peptide of interest 520 and the affinity region 522, the latter of which associates with the capture agent 516 following peptide synthesis.
  • the DNA is created as a double-stranded molecule.
  • the capture agent to this molecule, it can be tailed with terminal transferase to add a single-stranded oligo dT region to which a capture region associated with an oligo dA can hybridize.
  • FIG. 6 illustrates another general scheme for production of the arrays of the invention using single-stranded oligonucleotide arrays and a capture agent.
  • the method begins with oligonucleotides 600 arrayed on a surface 608, optionally attached to the surface via a linker sequence 606.
  • the oligonucleotides 600 each comprise a first region 614 that encodes an affinity tag and a stop codon, and which comprises a sequence that hybridizes selectively to a primer used in primer extension of the oligonucleotide, a coding region 612 encoding a peptide or complementary to a region encoding a peptide, and a second primer region 610 for attachment of a universal untranslated region 618 and/or a capture agent 616 comprising a capture agent that specifically binds to the affinity tag encoded by region 614.
  • Extension of the single-stranded molecule from primer region 614 and attachment of the untranslated region in step 611 produces a double-stranded template 602 that includes the capture agent positioned on the construct distal to the substrate surface 608.
  • the encoded region 612 encodes the peptide of interest
  • the primer extension region 614 encodes an affinity tag that binds to a capture agent 616, and a stop codon.
  • In vitro transcription and translation will produce the fusion peptide 650, comprising both the peptide of interest 620 and the affinity region 622, the latter of which associates with the peptide association moiety 616 following peptide synthesis.
  • FIG. 7 illustrates a more detailed method for constructing an array of the invention using attachment of oligonucleotides to separate surfaces, e.g., beads.
  • the arrays are constructed using oligonucleotide templates 700 comprising a region 710 for introduction of the capture agent, a region 712 encoding the polypeptide of interest, and a region 714 encoding an affinity tag followed by a stop codon.
  • the region 714 also comprises a primer site for primer extension of the single-stranded oligonucleotides, and it is optionally attached to the surface of the bead 718 by a linker molecule 708.
  • the oligonucleotides 700 on the beads 718 are provided in phosphorylated form, or alternatively phosphorylated in step 711 to allow addition of an oligonucleotide extension.
  • the oligonucleotide extension 702 is then ligated in step 713 to the template oligonucleotide 700 via a splint ligation technique using a ligation oligonucleotide 840 that selectively binds to both the oligonucleotide 800 and the oligonucleotide extension 802.
  • the oligonucleotide extension 702 comprises: a region 704 comprising an untranslated region and a primer region for use in splint ligation; optionally a linker molecule 738; and an oligonucleotide region 736 for attachment of the capture group 716.
  • the next step in production of the array is a combined step 715 to add the capture agent 716 and the primer 724 for primer extension.
  • a capture agent 716 associated with an oligonucleotide 742 that is complementary to region 736 of the oligonucleotide extension 702, and an oligonucleotide 724 to serve as a primer for primer extension are then hybridized to the oligonucleotides on the beads.
  • a primer extension step 717 is carried out to convert the single-stranded oligonucleotide encoding the peptide of interest and the affinity tag into a double- stranded template for use in in vitro transcription and translation.
  • In vitro transcription and translation is carried out in step 719 from this double-stranded template, creating a fusion peptide 750 comprising the peptide of interest 720 and an affinity tag 722.
  • the produced fusion peptide 750 is captured by the capture agent 716 as it diffuses from the oligonucleotide template region following translation.
  • the constructs that are synthesized directly on the beads may be used as synthesized, i.e., as an array of constructs on a collection of separate surfaces, e.g., beads, or they may be arrayed on a planar surface either before or after the synthesis of the oligonucleotide-peptide fusions on their surfaces.
  • measures are generally taken to prevent interaction of peptides with neighboring constructs, e.g., transcription and translation can be carried out with each bead type bearing a different sequence separated in different wells in a plate to ensure that peptides from a bead et comprising one construct are not captured by a different bead set.
  • FIG. 8 illustrates a more detailed, preferred method for constructing an array of the invention using attachment of oligonucleotides to a planar array.
  • the arrays are constructed using oligonucleotide templates 800 comprising a region 810 for introduction of the capture agent, a region 812 encoding the polypeptide of interest, and a region 814 encoding an affinity tag followed by a stop codon.
  • the region 814 also comprises a primer site for primer extension of the single- stranded oligonucleotides, and it is optionally attached to the surface of the substrate 804 by a linker molecule 808.
  • the oligonucleotides 800 on the planar arrays 804 are phosphorylated in step 811 to allow addition of an oligonucleotide extension.
  • the oligonucleotide extension 802 is then ligated in step 813 to the template oligonucleotide 800 via a splint ligation technique using a ligation oligonucleotide 840 that selectively binds to both the oligonucleotide 800 and the oligonucleotide extension 802.
  • the oligonucleotide extension 802 comprises: a region 804 comprising an untranslated region and a primer region for use in splint ligation; optionally a linker molecule 838; and an oligonucleotide region 836 for attachment of the capture group 816.
  • the next step in production of the array is a step 815 to add the capture agent 816 and the primer 824 for use in primer extension.
  • a capture agent 816 conjugated with an oligonucleotide 842 that is complementary to a region 836 of the oligonucleotide extension 802, and the primer 824 can be simultaneously or sequentially hybridized to the oligonucleotides on the slides.
  • a primer extension step 817 is carried out to convert the single-stranded oligonucleotide encoding the peptide of interest and the affinity tag into a double- stranded template for use in in vitro transcription and translation.
  • In vitro transcription and translation reactions in step 819 from this double-stranded template create a fusion peptide 850 comprising the peptide of interest 820 and an affinity tag 822.
  • the produced fusion peptide 850 is captured by the capture agent 816 as it diffuses from the oligonucleotide template region and away from the planar surface following translation.
  • the constructs of the invention are produced using a puromycin capture method.
  • FIG. 9 illustrates a general method for the construction of such arrays on a planar surface, although it is also envisioned that the arrays could be formed on beads, such as described in FIG. 7.
  • the method begins with double-stranded oligonucleotides 900 arrayed on a substrate surface 908, attached to the surface at either the 3' end or the 5' end of the oligonucleotide, optionally via a linker sequence 906.
  • the oligonucleotides 900 each comprise a coding region 912 encoding a peptide or of interest, a region comprising an RNA affinity tag followed by a stop codon 914, and an untranslated region 910 comprising a transcription promoter, to which an RNA binding region of DNA 918 and a puromycin 916 are attached.
  • An in vitro transcription step 911 produces an mRNA molecule comprising a region 926 encoding the peptide of interest and an affinity tag 924.
  • the affinity tag 924 selectively hybridizes to region 918 of the oligonucleotide, tethering the mRNA molecule to the construct. During translation, the ribosome will stall when it reaches the RNA-DNA hybridized region.
  • the peptide 920 is captured by the puromycin residue region 916.
  • a chemically reactive species e.g., an aldehyde tag
  • an aldehyde tag may be added to aid in the construction of the constructs and introduction of labeling elements of other binding regions.
  • introduction of a sulfatase consensus sequence recognized by the formylglycine-generating enzyme results in the site-specific introduction of aldehyde groups into recombinant proteins.
  • This consensus sequence can be between 6-13 amino acids, and the smallest such "aldehyde tags" are no larger than a His 6 tag.
  • Enzymatic modification at a sulfatase motif by formylglycine generating enzyme generates a formylglycine (FGly) residue, which allows site-specific attachment of a capture agent or other moiety of interest to the peptide by covalent capture on hydrazine or oxime labeled oligo templates.
  • FGE formylglycine generating enzyme
  • This modification is also reversible, and thus the introduction of this tag into recombinant peptides allows aldehyde- tagged proteins to be reversibly modified with multiple epitopes. Examples of aldehyde tags for use in the present invention are described in, e.g., US2008/0187956; T. Dierks and M.-A.
  • Fig. 10 the generation of covalently linked peptide arrays is shown in Fig. 10.
  • This approach is an exemplary method for carrying out the more general scheme outlined in Fig. 1 , but uses covalent attachment. It utilizes a method for the site-specific introduction of aldehyde groups into recombinant proteins using the 6-mer peptide consensus sequence (LCTPSP, termed as the aldehyde tag) recognized by the formylglycine-generating enzyme (FGE) which oxidizes a cysteine residue to formylglycine was recently reported.
  • an aldehyde tag is to add FGE to the in vitro transcription/translation process during peptide array production.
  • the peptides can be made with a C-terminal aldehyde tag, and the cysteine residue in the tag converted to the formylglycine residue that will react with an active group incorporated into the DNA templates on the array surface. This will result in an array of peptides that are covalently attached to their DNA templates.
  • Oligonucleotide templates encode the peptide sequences of interest 1006 and a region 1004 comprising an aldehyde affinity tag (LCTPSP) and a stop codon.
  • a universal sequence 1010 containing an untranslated region (UTR) with the T7 promoter and a ribosomal binding site (RBS) is added 1001 to the template via a distal tag 1018 and is used to extend the template to create a double-stranded nucleic acid 1002 comprising a capture group 1014 for the peptide of interest 1016 encoded by 1006 on the strand associated with the substrate 1008.
  • a reactive group 1022 is associated with the other strand of the double-stranded oligonucleotide template 1002, optionally on a linker molecule 1012.
  • the peptide comprising the peptide sequence of interest 1016 and the aldehyde tag 1020 is produced 1003, it is captured by interaction of the peptide of interest and the capture group 1014.
  • the aldehyde tag 1020 is converted 1005 to an aldehyde group 1024, and this interacts with reactive group 1022.
  • a single- stranded oligonucleotide encoding the peptide of interest 1016 remains associated to the peptide 1016 via the reactive group and the aldehyde group.
  • the capture agent 1014 can be removed.
  • peptides with sequences of interest 1116 are made via in vitro transcription/translation in the presence of FGE enzyme.
  • the aldehyde tag 1120 fused to the peptide of interest 1116 can be generated through interaction of a hydrazine or aminooxyacetic acid residue in 1116 with FGE, which converts 1101 the LCTPSR region incorporated in peptides to an aldehyde group 1124.
  • This aldehyde group is used to interact with the reactive group 1122 to associated the peptide of interest 1116 to the oligonucleotide encoding the peptide of interest 1106, the universal sequence and the sequence encoding the aldehyde tag 1104.
  • the coding sequences used for the production of the peptide arrays are provided on the surface of a flow cell, e.g., a flow cell used for sequencing techniques.
  • the coding sequences can be randomly arrayed on the surface of the flow cell.
  • the constructs of the arrays can be created by randomization of bases at defined positions during synthesis of an oligo template, by combining shorter oligos to form a longer template, or by deriving a library of sequences from a genomic DNA or cDNA library. Once the sequences have been placed on the surface of the flow cell, the identity of each sequence can be determined either before or after the production of the tethered protein and its use in a screening assay.
  • the coding sequences can optionally be clonally amplified in situ, with each sequence forming a 'cluster' of clonally amplified DNA molecules, prior to production of the peptide array on the surface.
  • very high packing densities can be achieved, e.g., several hundred million clusters can be arrayed on the flow cell surface.
  • the ability to array many millions of DNA templates and determine their sequence opens up possibilities of making large combinatorial protein libraries quite efficiently and inexpensively.
  • Oligonucleotides 1202 comprising the sequence of interest 1212, a linker region 1204 and a primer binding region 1214 are constructed on the flow cell surface 1208. Other linker regions on the surface 1206 are optionally present for the purposes of in situ amplification of the oligonucleotide template.
  • an oligonucleotide-antibody conjugate 1216 is annealed 1201 to the 3'-end of clustered DNA sequences. All DNA sequences code for peptides or proteins and have T7 promoter (or similar) in order to enable efficient transcription and an untranslated region (UTR) required for efficient translation.
  • T7 promoter or similar
  • the oligonucleotide portion of the conjugate 1216 is extended 1203 with DNA polymerase to create an oligonucleotide 1222 complementary to the attached oligonucleotide 1202.
  • a coupled transcription/translation reaction (TNT) 1205 leads to production of peptides or proteins 1220 encoded by clustered DNAs 1202 and their attachment to their own templates. All peptides have C-terminal affinity tag that is used for antibody capturing (not shown). Other capture molecule and affinity tag combinations can be used. Examples of such binding pairs include, but are not limited to, streptavidin and biotin, Ni-NTA and His6 tag, or two chemically reactive groups (aldehyde tag and hydrazine residue).
  • This approach is also applicable to other formats that permit in vitro cloning of single molecules.
  • a template molecule instead of capture and amplification on the surface of a flow cell using surface PCR, a template molecule can be captured on a bead and amplified by emulsion PCR. This process is used to generate clonal templates for certain next generation sequencing techniques. Although amplification of the individual templates is preferred, in certain aspects single molecule sequences can be determined without the use of clonal amplification.
  • the surface of the flow cell may comprise other linker regions on the surface that are present for the purposes of in situ amplification of the oligonucleotide template.
  • These constructs are sequenced either before construction of the peptide-nucleic acid hybrid or following construction and/or use (not shown). In the former case, the constructs are sequenced prior to production of these peptide-nucleic acid constructs, and an oligonucleotide- antibody conjugates are annealed to the 3 '-end of clustered DNA sequences on the surface.
  • the sequence can be determined following construction and/or identification of particular sequences in an assay format.
  • the antibodies can be loaded on the surface of the flow cell, coupled to the primer portion and directed only to the DNA clusters on the flow cell since the DNA-antibody complex will only hybridize to extended DNAs.
  • the antibodies coupled to oligonucleotides which are complementary to the oligos on the surface can introduced to the flow cell surface.
  • antibodies are loaded over entire surface of the flow cell since the DNA-antibody complexes will hybridize to oligonucleotide primers that are equally distributed over the surface of the flow cell. Peptide arrays are then formed from these DNA templates using one of the described methods herein.
  • Sequencing technology such as that provided by IlluminaTM Genome AnalyzerTM technology permits a DNA template to be sequenced from both ends in order to generate a 'paired end' pair of sequence reads.
  • this involves: a) obtaining the sequence of one end of the pair by sequencing one strand; then b) obtaining the sequence of the other end of the pair by sequencing the reverse complement strand.
  • Processes are carried out in order to generate and sequentially present the appropriate strand for sequencing. Therefore, arrays can also be constructed using the second (i.e. reverse complement) strand, simply by choosing the appropriate sequences to conjugate to the capture agent (i.e. in these examples, the antibody).
  • the arrays of the invention can utilize multiple ligation events to increase the length of the oligonucleotide template and thus the peptide produced through in vitro transcription and translation. This can be done through the use of restriction enzyme digestion and ligation, or preferably the use of splint ligation with primers that are complementary to both the oligonucleotide associated with the array and the oligonucleotide that is to be added to the array. For example, a region can be added to all or a subset of the constructs on an array using splint ligation such as that described for the addition of the oligonucleotide comprising the untranslated region.
  • a pool of oligonucleotides having two or more different sequences can be used with splint ligation primers having corresponding complementary sequences, allowing addition of multiple different variable oligonucleotides to the oligonucleotides associated with the substrate.
  • splint ligation primers having corresponding complementary sequences allowing addition of multiple different variable oligonucleotides to the oligonucleotides associated with the substrate.
  • NCAM Neural Cell Adhesion Molecule
  • the ability to create longer oligonucleotide templates on the arrays of the invention can provide tools to better elucidate the activity and protein interaction of the various forms of proteins, and give insight into regulation, e.g., for therapeutic development.
  • Numerous other such uses of constructed regions having certain constant domains, e.g., the N- terminus, the C-terminus, active binding sites, enzymatic active regions, etc. are envisioned with the arrays of the invention, as will become clear to one skilled in the art upon reading the present disclosure.
  • the arrays can also be utilized two or more times.
  • the binding of the peptide to the oligonucleotide via the affinity tag-capture agent interaction is a much tighter bond than that seen in most transient protein-protein interactions, and thus many proteins and compounds used to interrogate the arrays can be effectively removed to allow the array to be used in other interrogations.
  • the array can be regenerated by removal of all hybridized portions of the constructs, effectively renewing the initial array used for construction of the peptide array.
  • the single- stranded oligonucleotides can be again converted to a double-stranded oligonucleotide, and the remaining steps of the methods of the invention used to recreate a peptide comprising array of the invention.
  • the initial oligonucleotide is a single-stranded oligonucleotide that is synthesized on the substrate surface and converted to a double-stranded molecule through, e.g., primer extension.
  • Primer extension can be initiated by hybridization of a primer to a primer-binding region on the single- stranded oligonucleotide template where the result after extension is a double- stranded template including the untranslated region, the peptide coding region and the region encoding the affinity tag.
  • the primer-binding region incorporates the affinity tag sequence and/or the stop codon sequence, although the primer-binding region may also be located 3 ' to the affinity tag sequence and the stop codon.
  • the primer extension process typically utilizes a one or more primers complementary to the oligonucleotide template, which drives synthesis of the opposite strand in the presence of a polymerase and free dNTPs.
  • a polymerase stop feature introduced into the DNA molecule following the extension of the oligonucleotide template region.
  • This feature may include a synthetic linker, such as a polyethylene glycol (PEG) feature, a nucleotide analog that cannot be used by the polymerase, a nick introduced onto a double-stranded primer region upstream of the template region ⁇ e.g., by a nickase or the degradation of uracil) and the like.
  • PEG polyethylene glycol
  • primers/ affinity tag encoding regions may be used in a single construct, which may allow a single oligonucleotide to be used with various affinity tag-capture agent binding pairs in construction of the array.
  • the constructs may also have such sequences distal from the substrate surface to aid in selective ligation of capture agents. Substrates for Use in the Invention
  • substrates of the invention are nonporous, particularly when random arrays of single molecules are analyzed by hybridization reactions requiring small volumes.
  • Suitable substrates include substrates composed of materials such as glass, polyacrylamide-coated glass, ceramics, silica, silicon, quartz, various plastics, and the like.
  • the substrate is a bead.
  • the substrate is a planar surface.
  • the planar surface is in the range of from 0.02 to 20 cm or even larger.
  • the limit on substrate size is based on the detection methods used and the ability to resolve (e.g., in the case of fluorescent markers, the ability to optically resolve) the different constructs and/or regions of constructs on the surface. As detection methods continue to improve, substrate size may increase and/or the density of the constructs on a substrate may increase.
  • the format of the substrates of the present invention includes substantially planar surfaces as well as substrates with introduced variations to the substrate surface, e.g., depressions, wells, pedestals and the like.
  • Such substrates are generally comprised of a material or group of materials having a rigid or semirigid surface or surfaces.
  • Such substrates can be produced, e.g., using multi-layer coating technologies or other well known techniques in the art.
  • Examples of techniques for production of pattered arrays includes thermal and/or electron beam vapor deposition, replication, transfer, or film deposition; the CVD-type processes (LPCVD, PECVD etc.); PVD-type processes such as sputtering, e.g., DC magnetron sputtering; ion-assisted deposition processes and sol-gel processes. Layers of substrate are optionally transferred onto the base by bonding or molecular adhesion.
  • linkers may be used to attach the array constructs to a surface.
  • Numerous types of linkers can be used, and the linker will generally be selected based on the type of construct, (amino acid, nucleic acid, etc.), the desired properties of the linker (length, flexibility) and other similar characteristics.
  • Such linkers may comprise nucleotides, polypeptides, or a suitable synthetic material.
  • the linker structures are preferably hydrocarbon base polymers which are comprised of biocompatible polymeric materials ⁇ e.g., polyethylene glycol). Also, the choice of linker depends upon whether the capture agent is associated with the construct at the linker portion of the construct.
  • the surface-immobilized constructs comprise a cleavable linker directly attached to the substrate that allows specific constructs to be separated from the substrate.
  • the cleavable linker will be the same or identical for all of the surface-immobilized constructs.
  • certain subsets of constructs on the substrate will have the same cleavable linker, where this cleavable linker differs from the cleavable linkers used with the other subsets on the same substrate surface. This allows certain constructs to be separated from the substrate when others are not.
  • a microfluidic gasketing system is used on the substrate surface to effectively isolate each feature on the peptide array to enable control of diffusion during synthesis of the array and analysis using the array. This allows the reaction conditions to be better controlled in one or more portions of the array. For example, the in vitro transcription and translation can be carried out for a longer period because the containment system limits diffusion. Also, once peptides are captured, washing and/or reagent exchange steps can be carried out, enabling new reactions to take place, such as covalent linkage of the peptide. Finally, the containment system provided by gasketing allows peptides to be captured on a second surface. In this way, multiple peptide arrays can be produced from an initial template DNA array without the requirement of diffusion through a membrane, such as required in He M Nat. Methods 5:175-177 (2008).
  • binding pairs can be used to design the affinity tags and capture agents used in the arrays of the invention. These include, but are not limited to, streptavidin and short streptavidin binding peptides such as StrepTag (Schmidt et al, 1996; Schmidt & Skerra, 1994; Skerra & Schmidt, 2000), StrepTag II (Schmidt & Skerra, 2007; Voss & Skerra, 1997), and HPQ motifs (Giebel et al, 1995; Helms et al, 2007); oligo histidine peptide tags and His6 binding groups (Crowe et al, 1994; Smith et al, 1988); FLAG peptide tags and His6 peptide group; biotin and streptavidin, biotin and avidin, and antibody-antigen pair, and the like.
  • streptavidin and short streptavidin binding peptides such as StrepTag (Schmidt et al, 1996; Schmidt & Skerra, 1994; Skerra & Schmidt
  • the strength of the interaction of a peptide binding pair can be characterized by its "binding affinity" of one part of the binding pair to a given binding site or epitope on the other member of the binding pair.
  • binding affinity of one part of the binding pair to a given binding site or epitope on the other member of the binding pair.
  • antibodies are characterized by their "binding affinity" to a given binding site or epitope. Every antibody is comprised of a particular 3- dimensional structure of amino acids, which binds to another structure referred to as an epitope or antigen.
  • the selective binding of a binding partner to a composition is a simple bimolecular, reversible reaction, not unlike the binding of an antibody to its antigen.
  • the reaction can be analyzed by standard kinetic theory. Assuming a single binding site the reaction is represented by the equation I as follows:
  • brackets denote concentration in moles per liter or liters per mole.
  • a typical value for the binding affinity K a which is also referred to as "K” and is the "affinity constant" which for a typical antibody is in a range of from about 10 5 to about 10 11 liters per mole.
  • the K a is the concentration of free antigen needed to fill half the binding sites of the antibody present in solution with the antigen. If measured in liters per mole a higher K a (e.g. 10 11 ) or higher affinity constant indicates a large volume of solvent, a very dilute concentration of free antigen, and as such indicates the antibody has a high binding affinity for the epitope.
  • K a is measured in moles per liter a low K a (e.g. 10e 5 ) indicates a less concentrated solution of the free antigen needed to occupy half of the antibody binding sites, and as such a high binding affinity.
  • Equation III the units for K are liters per mole. Typical values in liters per mole are in a range of from about 10 5 to about 10 11 liters per mole.
  • K [Ag] where the units for K are in moles per liter, and the typical values are in a range of 10 "6 to 10 "12 moles per liter.
  • typical binding affinities can vary over six orders of magnitude.
  • what might be considered a useful antibody might have 100,000 times greater binding affinity as compared to the binding affinity of what might be considered a different antibody, which is also considered useful.
  • the resolvability of the constructs is enhanced by providing one or more features on the array that are "empty", or without a construct or capture agent, providing additional space that prevents excessive diffusion of a peptide from its construct to neighboring constructs.
  • Peptide diffusion following in vitro translation is more easily controllable over relatively short distances (e.g., 75-225 ⁇ ), and having empty features between constructs, and preferably between 0 to 2 empty features, allows the resolvable and reproducible identification of individual constructs, thus allowing the identification of binding of an agent of interest to a specific peptide on the array.
  • arrays having individual constructs comprising the same capture agent FIG. 13
  • FIG. 13 A may have one empty feature 1304 between the coding constructs 1302.
  • two or more empty features can be used between constructs.
  • FIG. 13C illustrates an array having two empty features 1304 between coding constructs 1302 having the same capture agent (FIG. 13C).
  • each of the coding constructs 1302 encode the same affinity tag, which specifically binds to the capture agent.
  • multiple capture agents and affinity tags can be used for with the individual constructs, and constructs comprising the different capture agent-affinity tag pairs are interspersed on the array in a configuration to prevent diffusion and inadvertent capture of a peptide created from a construct by its neighboring constructs.
  • non-specific signal due to diffusion of approximately 150 ⁇ or more at a density of 100% can be prevented by using four sets of constructs (1302, 1306, 1308 and 1310), each set having a different affinity tag - capture agent pair.
  • nonspecific signal due to diffusion of approximately 225 ⁇ or more as shown ion FIG.
  • 13D can be prevented by using nine sets of constructs (1302, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320), each set having a different affinity tag - capture agent pair. Even though diffusion will still take place, the diffusing peptides will not be captured at adjacent features because these will have incompatible capture agents.
  • capture agent-affinity tag pairs enable the use of multiple constructs having the same peptide of interest, each associated with a capture agent targeting a subset of the constructs on the array. This allows the same peptide of interest to be represented on the array with different capture agent- affinity tag pairs. For example, some peptides of interest may be incompatible with certain peptide affinity tags.
  • capture agent- affinity tag pairs allows the association of a peptide of interest with more than one such pair, which will to mitigate such effects by representing peptides with two different affinity tags rather than just one.
  • the diffusion of a peptide to its surrounding constructs can be used as an advantage in determining positive results on an array.
  • the small amount of peptide diffusion between constructs having the same capture agent can actually be used to confirm a positive binding result at a particular construct, as the small amount of diffusion to surrounding constructs provide a "halo" effect of binding, and thus an identifiable but lesser signal on the constructs surrounding the one to which an agent selectively binds.
  • FIG 14 illustrates an array having a construct 1402 which selectively binds to an agent and which registers as a positive result in a binding assay.
  • the array also has numerous constructs having no signal 1404 and certain other constructs that spuriously register as false positives.
  • the surrounding constructs 1406 Due to peptide diffusion from the construct 1402 to the surrounding constructs 1406, the surrounding constructs 1406 show a lesser but detectable binding as compared to the true positive construct 1402. The difference in binding between the positive construct 1402 and its neighbors 1406 is statistically significant, thus still allowing identification of the true positive 1402. This halo effect differentiates a binding activity on a construct from other potential spurious sources of a positive result, such as shown in constructs 1408, serving as an internal control mechanism to reduce the number of false positives in an assay.
  • oligonucleotides Single-stranded oligonucleotides were used for the construction of the arrays.
  • the initial oligonucleotides were 60-mers comprising common regions: a region encoding an affinity tag, either a FLAG peptide (DYKDDDDK)(SEQ ID NO: l) or its shorter version FLAGS (DDDDK) at the 3 '-end; a region encoding a peptide of interest, either an HA peptide (YPYDVPDYA)(SEQ ID NO:2) or an AU1 peptide (DTYRYIDYA)(SEQ ID NO:3); and a primer region for the attachment of a universal untranslated region.
  • the primer region was designed to allow for the addition of a long untranslated region comprising a T7 promoter region at the 5'-end followed by a ribosomal binding site (RBS).
  • Oligonucleotides were synthesized on an Expedite 8909 DNA synthesizer, using formylindole phosphoramidite (Glen Research, Sterling, VA) to introduce an aldehyde group at either the 5'- or 3 '-terminus of the oligonucleotides.
  • 3 '-end modification the synthesis was started on dT-CPG support resulting in a Formylindol-dT residue at the 3 '-end of all oligonucleotides.
  • Internal Cy5 modifier phosphoramidite (Glen Research) was used to introduce Cy5 dye at the 3 '-end of oligonucleotides. In this case, synthesis was started on dA-CPG support resulting in a Cy5-dA residue at the 3'-end of oligonucleotides.
  • Example 2 Production of Arrays on Beads
  • a method for synthesizing arrays of the invention on amino-modified silica beads using established protocols was developed. First, amino groups were incorporated into 3 um silica beads (Bangs Labs) by treatment with 0.5% 3- aminopropylthriethoxysilane solution in ethanol (Aldrich). Next, the beads containing amino groups were treated with 0.1M cyanuric chloride solution (Aldrich) containing 0.2M diisopropylethylamine in acetonitrile followed by treatment with 2% hydrazine (Aldrich) solution in DMF. Washing with ethanol, acetonitrile and DMF was carried out between each step respectively.
  • the oligonucleotides covalently linked to the beads comprise a primer region for introduction of the capture agent, an untranslated region comprising a transcriptional start site and an RBS, a region encoding either the HA or the AU1 peptide of interest, and a region encoding a FLAG affinity tag followed by a stop codon.
  • the FLAG region also comprises a primer site for primer extension of the single-stranded oligonucleotides.
  • the oligonucleotides comprise a 5' phosphate to allow ligation of the oligonucleotide extensions.
  • the beads were concentrated as a pellet using a conventional benchtop centrifuge, and the liquid was carefully aspirated so as not to disturb the pellet.
  • the beads were placed in 0.2 ml PCR tubes. 50 ⁇ of 1XPBST [3mM NaH 2 P0 4 , ImM KH 2 P0 4 , 150mM NaCl, 0.05% Tween 20 (pH 7.4)] was added and the beads were washed once.
  • the 1XPBST was removed, 50 ⁇ of KB solution [50 mM Tris-HCl, 10 mM MgCl 2 (pH 7.5)] solution was added and the beads were washed once in this buffer.
  • oligonucleotide extension comprising an untranslated region and region for use in splint ligation, a PEG linker molecule; and a a T30 oligonucleotide for attachment of the capture group was ligated to the oligonucleotide linked to the bead using a splint primer that hybridizes to both the substrate-linked oligonucleotide and the extension primer.
  • a ligation mixture comprising 37.5 ⁇ H 2 0, 5 ⁇ ⁇ T4 DNA Ligase Buffer (NEB), 5 ⁇ of preannealed extension and splint oligonucleotides (10 ⁇ ) and 7.5 ⁇ T4 DNA Ligase (400,000 U/ml, NEB) was added to each tube, and incubated for 30 minutes at room temperature in a rotator.
  • NEB DNA Ligase Buffer
  • the beads were treated to denature the splint ligation primer from the ligated oligonucleotide extension and template oligonucleotide linked to the bead.
  • the beads were washed three times with 50 ⁇ 1XPBST.
  • the beads were treated with 50 ⁇ 95% aq Formamide and 1 mM EDTA, incubated five minutes at room temperature, and this treatment was repeated an additional time.
  • the beads were then washed three times with 50 ⁇ 1XPBST.
  • An anti-FLAG antibody capture agent conjugated with an oligonucleotide that is complementary to a region of the oligonucleotide extension, and an oligonucleotide to serve as a primer for primer extension were then hybridized to the oligonucleotides on the beads.
  • the beads were washed once with 50 ⁇ lXHyb Buffer [450 mM NaCl, 30 mM sodium dihydrogenphosphate, 13mM EDTA, 0.025% Tween-20 (pH 7.4)].
  • a capture-agent primer solution [22.5 ⁇ H 2 0, 25 ⁇ 2X Hyb Buffer, 0.5 ⁇ extension primer ( ⁇ ; TTACTTATCGTCGTCGTCXSEQ ID NO:4), 2 ⁇ capture agent (A30-Cy5-anti- FLAG -IgG ⁇ 6.7 ⁇ )] was added to the beads, and incubated at room temperature for 30 minutes in a rotator.
  • Primer extension was carried out to convert the single-stranded oligonucleotide encoding the peptide of interest and the FLAG affinity tag into a double-stranded, bead linked oligonucleotide.
  • the beads with the primer extension primer hybridized to the bead-linked oligonucleotide were washed twice with 50 ⁇ of lXHyb Buffer, and once with 50 ⁇ lXReactll [50mM Tris HC1 , lOmM MgC12, 50mM NaCl, (pH 8)].
  • PURExpress solution 70 ⁇ H20, 125 ⁇ Solution A, 50 ⁇ Solution B, , 5 ⁇ 8 U/ ⁇ Murine RNase Inhibitor, NEB
  • PURExpress solution 70 ⁇ H20, 125 ⁇ Solution A, 50 ⁇ Solution B, , 5 ⁇ 8 U/ ⁇ Murine RNase Inhibitor, NEB
  • the peptides of interest were detected on the beads by primary antibodies that recognize an epitope on the respective peptides of interest.
  • the primary antibody was used at a concentration of 67 nM, and the beads were incubated in the presence of the primary antibody for 30 minutes at room temperature. Incubation was performed with a labeled secondary antibody that binds specifically to the primary antibody at a concentration of 17nM.
  • the beads were imaged using a DM6000B automated fluorescence microscope and imaging system.
  • the arrays were constructed using oligonucleotides comprising a primer region for introduction of the capture agent, a region encoding an AU1 (DTYRYIDYAXSEQ ID NO:5), AU5 (TDFYLKDYA)(SEQ ID NO:6), HA (YPYDVPDYAXSEQ ID NO:7), or V5 (IPNPLLGLD)(SEQ ID NO: 8) 9-mer peptide, and a region encoding a FLAG affinity tag followed by a stop codon.
  • the region also comprised a primer site for hybridization of a primer to be used for primer extension of the single-stranded oligonucleotides.
  • the oligonucleotide templates were covalently attached to the activated slide surfaces via a 3 '-aldehyde linkage.
  • 30 ⁇ of 200 ⁇ 5'-aldehyde oligonucleotide 3 in 100 mM Na- citrate buffer, pH 5.1, 1.5M NaCl was placed between two reporter slides. The reaction was allowed to proceed for 18 hours at room temperature in a humidified chamber. The slides were washed three times two times with water, dried and refrigerated until used.
  • oligonucleotide slides were then prepared for array construction in a flow through apparatus by the addition of 150 ⁇ of 100% Formamide.
  • 250 ⁇ of 1XPBST was added in two 125 ⁇ aliquots to each slide and incubated for 1 minuteand then this wash step was repeated.
  • 250 ⁇ of Blocking solution [IX Hyb Buffer, 5x Denhardt s Solution, and 0.1 mg/ml Denatured Salmon Sperm DNA] was added to each slide in two 125 ⁇ aliquots, and incubated for 5 minutes.
  • 250 ⁇ fresh Blocking solution was added and the slides were incubated for 10 minutes.
  • the oligonucleotide extension comprising an untranslated region and a T30 region for use in splint ligation, a PEG linker molecule, and an oligonucleotide for attachment of the capture group was ligated to the oligonucleotide template using a splint primer, which hybridizes to both the substrate-bound oligonucleotide and the oligonucleotide extension.
  • a splint primer which hybridizes to both the substrate-bound oligonucleotide and the oligonucleotide extension.
  • 450 ⁇ (150 ⁇ , 3 times) of KB solution was then added to the slides in 3 equal aliquots, and the slides were incubated for 30 seconds at room temperature. This wash was repeated two times, and the flow-through chambers comprising the slides were placed in the humidified incubation chamber pre-equilibrated to 4°C in the refrigerator and incubated for 10 minutes.
  • a ligation mixture comprising 112.5 ⁇ H 2 0, 15 ⁇ lOx T4 DNA Ligase Buffer (NEB), 7.5 ⁇ 20 ⁇ / ⁇ 1 extension oligonucleotides and 2.5 ⁇ T4 DNA Ligase (400,000 U/ml, NEB) was added to each slide, and the slides were incubated for 30 minutes at room temperature. An additional 150 ⁇ aliquot of cold ligation mixture was then added, and the slides were incubated for an additional 30 minutes at room temperature. The resulting structures comprised the oligonucleotide template ligated to the extension oligonucleotide.
  • the slides were treated to denature the splint ligation primer from the ligated substrate-linked, extended oligonucleotide.
  • the slides were incubated with 450 ⁇ of KB solution [50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 ] added to each slide in 3 equal aliquots, for 30 seconds at room temperature.
  • the slides were then loaded with 450 ⁇ 95% aq. Formamide with 1 mM EDTA, added in 3 equal aliquots, and incubated for 1 minute. This was replaced with another 450 ⁇ 95% aq. Formamide with 1 mM EDTA, added in 3 equal aliquots, and incubated for an additional 5 minutes.
  • An anti-FLAG antibody capture agent conjugated with an oligonucleotide that is complementary to region of the extension oligonucleotide, and an oligonucleotide to serve as a primer for primer extension were then hybridized to the oligonucleotide s on the slides.
  • 450 ⁇ lXHyb Buffer was introduced to each slide, and incubated for 30 seconds at room temperature. This was repeated four times.
  • a capture-agent-primer solution [67.5 ⁇ H 2 0, 75 ⁇ 2X Hyb Buffer, 1.5 ⁇ extension primer ( ⁇ ; TTACTTATCGTCGTCGTC)(SEQ ID NO:9), 6 ⁇ capture agent (A30Cy5-anti-FLAG-IgG ⁇ 6.7 ⁇ )] was added to the flow through chamber, and it was incubated in a humidified chamber at room temperature for 30 minutes.
  • the slides were treated to denature the splint ligation primer from the ligated substrate-linked, extended oligonucleotide.
  • the slides were incubated with 450 ⁇ of KB solution [50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 ] added to each slide in 3 equal aliquots, for 30 seconds at room temperature.
  • the slides were then loaded with 450 ⁇ 95% aq. Formamide with 1 mM EDTA, added in 3 equal aliquots, and incubated for 1 minute. This was replaced with another 450 ⁇ 95% aq. Formamide with 1 mM EDTA, added in 3 equal aliquots, and incubated for an additional 5 minutes.
  • An anti-FLAG antibody capture agent conjugated with an oligonucleotide that is complementary to region of the extension oligonucleotide, and an oligonucleotide to serve as a primer for primer extension were then hybridized to the oligonucleotides on the slides.
  • 450 ⁇ lXHyb Buffer was introduced to each slide, and incubated for 30 seconds at room temperature. This was repeated four times.
  • a capture-agent-primer solution [67.5 ⁇ H 2 0, 75 ⁇ 2X Hyb Buffer, 1.5 ⁇ extension primer ( ⁇ ; TTACTTATCGTCGTCGTC)(SEQ ID NO: 10), 6 ⁇ capture agent (A30Cy5-anti-FLAG-IgG ⁇ 6.7 ⁇ )] was added to the flow through chamber, and it was incubated in a humidified chamber at room temperature for 30 minutes.
  • Primer extension was carried out to convert the substrate-linked, single- stranded oligonucleotide encoding the peptide of interest and the FLAG affinity tag into a double-stranded substrate-linked molecules.
  • the slides with the primer extension primer hybridized to the slide-linked oligonucleotides were treated with 450 ⁇ of lXHyb Buffer, added in three equal aliquots, incubated for 30 seconds, and then twice treated with 450 ⁇ lXReactll and incubated for 30 seconds.
  • 150 ⁇ of an extension mixture [105 ⁇ H 2 0 , 15 ⁇ ⁇ React II, 15 ⁇ 25mM dNTPs, 15 ⁇ 2 U/ ⁇ Diluted DNA Pol I (Large Fragment, (Invitrogen, Carlsbad, CA)) was added and the slides were incubated in a humidified incubation chamber pre- equilibrated to 37°C for 45 minutes.
  • PURExpress solution 70 ⁇ H20, 125 ⁇ Solution A, 50 ⁇ Solution B, 5 ⁇ 8 U/ ⁇ Murine RNase Inhibitor, NEB
  • the flow-through chambers were placed in a humidified incubation chamber pre- equilibrated to 37°C, and incubated for between 0.5 - 60 minutes at 37°C.
  • the peptides of interest were detected on the array surface by primary antibodies that recognize an epitope on the peptide of interest. The primary antibody was added at a concentration of 6.7 nM (in Superblock, ThermoSci), and the slide was incubated for 15 minutes at room temperature. This step was then repeated.
  • the AU1, AU5, HA and V5 9-mer peptides were constructed on three microscope slides in the same layout, with three spots of each peptide per slide, and detected specific signal by treating each slides with a specific antibody for the AU1 , AU5, or HA peptides.
  • the V5 peptide provided a negative control for all three detection conditions.
  • the peptides were selectively detected by the appropriate antibody: three AU1 peptides were detected with the anti-AUl antibody (16A), three AU5 peptides were detected with the anti-AU5 antibody (16B), and three HA peptides were detected with the anti-HA antibody (16C).

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Abstract

La présente invention concerne des matrices, des procédés de construction de matrices, et des procédés d'utilisation de telles matrices. Les matrices de l'invention comprennent un substrat avec deux constructions discrètes ou plus ou des ensembles discrets de constructions associés sur la surface du substrat, facultativement par l'intermédiaire d'une molécule de lieur. Les constructions comprennent un oligonucléotide comprenant une région codant pour un peptide d'intérêt et une étiquette d'affinité et une région non traduite, un peptide de fusion comprenant à la fois le peptide d'intérêt et l'étiquette d'affinité et un agent de capture qui forme une paire de liaison avec l'étiquette d'affinité.
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CN102648294A (zh) 2012-08-22
EP2510127A4 (fr) 2013-03-13
EP2510127A1 (fr) 2012-10-17
US20120270748A1 (en) 2012-10-25
EP2510127B1 (fr) 2015-06-10
AU2010328226A1 (en) 2012-06-28

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