WO2018005720A1 - Procédé de détermination de la liaison moléculaire entre des banques de molécules - Google Patents

Procédé de détermination de la liaison moléculaire entre des banques de molécules Download PDF

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WO2018005720A1
WO2018005720A1 PCT/US2017/039862 US2017039862W WO2018005720A1 WO 2018005720 A1 WO2018005720 A1 WO 2018005720A1 US 2017039862 W US2017039862 W US 2017039862W WO 2018005720 A1 WO2018005720 A1 WO 2018005720A1
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protein
mrna
target
binder
sequence
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Kettner John Frederick GRISWOLD
Neng FANG
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President And Fellows Of Harvard College
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    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

  • the present invention relates in general to methods of determining the molecular binding between libraries of molecules.
  • Protein display methods are typically employed for purposes of affinity maturation of proteins.
  • affinity maturation schemes a library of proteins are displayed, and passed over a surface saturated with excess copies of a single target molecule. Proteins that remain attached to the target molecule after washing are subsequently amplified and diversified. Over multiple rounds of affinity maturation processes, high affinity proteins for the target molecule are enriched.
  • affinity maturation and enrichment processes are typically limited to singular targets due to diffi culties to precisely assign interactions between libraries of affinity matured binder molecules and libraries of target molecules.
  • the disclosure provides methods of determining the molecular binding between libraries of molecules.
  • the disclosure provides methods for the determination of molecular interactions from the pairing of nucleic acid barcodes following molecule-molecule binding events.
  • the disclosure pro vides methods for retrieval of nucleic acid barcode sequences encoding the pair of binding molecules.
  • the disclosure provides a method for determining the molecular binding between a target molecule library and a binder molecule library including the steps of: (a) contacting the target molecule library comprising a plurality of mRNA -protein conjugates with the binder molecule library comprising a plurality of mRNA-protein conjugates under conditions where one or more proteins in the target library bind to one or more proteins in the binder library, wherein the mRNA of each mRNA-protein conjugate comprises sequences of an open reading frame (ORF) encoding the conjugated protein and a 3' untranslated region (3' UTR) sequence motif comprising a barcode sequence, (b) crossimking the bound target-binder protein pairs to form protein-protein complexes, (c) transferring the cross-linked protein- protein complexes of the target-binder protein pairs with their respective mRNAs conjugated thereto into a gel support, and (d) determining the molecular binding of the target and binder protein pairs by determining the sequences of the m
  • the disclosure provides that the plurality of mRNA-protein conjugates of the target library are immobilized to a solid support.
  • the disclosure provides that the plurality of mRNA -protein conjugates of the target library are immobilized to a solid support via a protein immobilization tag.
  • the disclosure provides that the protein immobilization tag is fused with the protein of the mRNA-protein conjugate of the target library.
  • the disclosure provides that the protein immobilization tag forms a covalent bond to a surface attachment ligand on the solid support.
  • the disclosure provides that the surface attachment ligand is cleavable from the solid support.
  • each DNA molecule comprises a sequence of an open reading frame (ORF) encoding the conjugated protein of the target library or the binder library and a sequence encoding the 3' untranslated region (3' UTR) sequence motif.
  • ORF open reading frame
  • the 3' UTR sequence motif of the DNA molecule for the target library comprises sequences encoding a polypeptide linker, polystop codons, PCR priming sequences, isothermal PCR priming sequences, an enzyme recognition site, a barcode sequence, and DNA scaffold sequences for in vitro transcription, translation and mRNA display.
  • the disclosure provides that the 3 ' UTR sequence motif of the DNA molecule for the binder library comprises sequences encoding a polypeptide linker, polystop codons, PCR priming sequences, isothermal PCR priming sequences, an enzyme recognition site, a unique molecule identifier sequence (UMI), a barcode sequence, and DNA scaffold sequences for in vitro transcription, translation and mRNA display.
  • UMI unique molecule identifier sequence
  • the disclosure provides that a puromycin oligo is attached to the 3' UTR of the in vitro transcribed mRNA for mRNA display.
  • the disclosure further provides degrading non-attached mRNA by a 3' to 5' directed riboexonuclease after the attachment step.
  • the disclosure provides that the riboexonuclease is R ase R, exoribonuclease II, or exonuclease T.
  • the disclosure provides that the attaching is via UV crosslinking or ligation.
  • the disclosure provides that the mRNA is covalently attached to the C-terminus of the encoded protein.
  • the disclosure provides that the mRNA-protein conjugates are passivated by reverse transcription of the mRNAs to form an mRNA-cDNA duplex prior to the contacting step.
  • the disclosure provides further cleaving the cross-linked protein-protein complexes of the target-binder protein pairs from the solid support.
  • the disclosure provides further casting the cross-linked protein-protein complexes of the target-binder protein pairs in a gel.
  • the disclosure provides further in gel PCR amplification of the mRNA-cDNA duplex conjugated to the target-binder protein pairs.
  • the disclosure provides that the amplified PCR products are sequenced through the ORF and barcode regions to determine the molecular identity of the target-binder protein pairs.
  • the disclosure provides that the barcode sequence is unique to the ORF within the mRNA.
  • the disclosure provides that further comprising in gel PCR amplification of the unique barcoded region in the mRN A-cDNA duplex conjugated to the target-binder protein pairs.
  • the disclosure provides that the PCR ampiicons are joined.
  • the disclosure provides that the joining is carried out via restriction enzyme digestion of the enzyme recognition site and ligation.
  • the disclosure provides that the restriction enzyme is a type IIS endonuclease.
  • the disclosure provides that the joining is carried out via
  • the disclosure provides that the joining is carried out via overlap extension PCR.
  • the disclosure provides that the molecular identity of the target-binder protein pairs of the protein-protein complexes is determined by sequencing the unique barcoded regions in joined PCR ampiicons.
  • the disclosure provides that the barcode sequence is an orthogonal PCR priming sequence.
  • the disclosure provides that the barcode sequence is a set of mutually orthogonal 20 base pair PCR priming sequence.
  • the enzyme recognition site is a type US endonuclease recognition site.
  • the disclosure provides that the in vitro transcription and translation are carried out in prokaryotic or eukaryotic systems.
  • the disclosure provides that the joined PCR amplicons are further amplified for sequencing and clonal isolation of the desired binder.
  • the disclosure provides that the clonal isolation of the desired binder is carried out first by selecting all the binders that bind to a target, and second by selecting a single binder that binds to the target.
  • the disclosure provides that the steps can be repeated to enrich for affinity maturation of a target molecule against a plurality of diversified binder molecules.
  • the disclosure provides that the ORF within the mRNA can be barcoded in either the 5' UTR or 3' UTR.
  • the disclosure provides that the method can be used for determining the molecular binding of DNA barcoded chemical libraries.
  • FIGS. 1A and IB depict schematics of mRNA display.
  • FIG. 1 A depicts a normal translation termination scheme.
  • FIG. IB depicts an exemplary mRNA display scheme where the ribosome translates puromycin linked mRNA without release factors 1, 2 and 3.
  • FIG. 2 depicts a schematic of preparation of UT ' R barcoded ORFeome libraries using Gibson assembly .
  • FIG. 3 depicts a cloning schematic of barcoded ORFeome transfer to gBlock.
  • FIG. 4 depicts schematics of tagging Illumina PGR priming sequences to barcoded amplicon sequences for next, generation sequencing (NGS) determination and verification of the barcoded amplicon sequences.
  • NGS generation sequencing
  • FIG. 5 depicts schematics of a target-binder pair immobilized on a solid support.
  • the target and binder are each conjugated to its encoding mRNA, which can be passivated as an mRNA-cDNA duplex.
  • the target molecule is immobilized to the solid support via a protein immobilization tag.
  • the protein immobilization tag can be covalently linked to a ligand on the surface of the solid support.
  • FIG. 6 depicts schematics of stabilizing the protein-protein complexes bound to the solid support via crosslinking.
  • FIG. 7 depicts schematics of gel casting of the crosslinked protein-protein complexes which are cleaved off of the solid support and polony amplification of the barcoded mRNAs conjugated to the proteins.
  • FIG. 8 depicts schematics of in gel polony amplification of colocalized nucleic acids.
  • FIG. 9 depicts schematics of covalent joining of colocalized nucleic acid polonies by Golden Gate i gel reactions.
  • FIG. 10 depicts schematics of in gel PCR and elution of amplicon DNA containing paired barcodes for NGS library preparation
  • FIG. 11 depicts schematics of interactome map recovery from NGS data.
  • FIG. 12 depicts schematics of clonal recovery procedure for binder species against each target.
  • the present disclosure is directed to methods of determining the molecular binding between libraries of molecules.
  • the disclosure provides methods for affinity maturation between libraries of molecules. Affinity maturation generally refers to a process where the binding affinity between a target and a binder molecule is increased by modification of the binder molecule.
  • the disclosure provides methods for multiplexing affinity maturation of one or more molecule libraries, such as protein target libraries against one or more molecule libraries, such as binder libraries.
  • the target and binder molecules are nucleic acid barcoded proteins, polypeptides or small molecules.
  • determining the interaction between the target and binder molecule pairs is by determining the sequence of the barcoded nucleic acids associated with the respective target and binder molecules.
  • affinity maturation of protein target libraries against binder libraries requires three fundamental functionalities including 1) an affinity maturation process, 2) the ability to determine all pairwise interactions between and within protein libraries and 3) retrieval of protein binder pairs between and within diverse libraries according to interaction data.
  • the disclosure provides methods that in addition to facilitating library affinity maturation, enable the determination of pairwise molecular interactions by DNA sequencing. Further, the disclosure provides methods that facilitate retrieval and recovery of populations of individual binding pairs from the pool of the molecule libraries.
  • the disclosure provides methods for affinity maturation of molecules such as proteins.
  • Methods for attaching genetic information in the form of nucleic acids to protein sequences have been described in the art as protein display methods.
  • Protein display methods are typically employed for purposes of affinity maturation of proteins.
  • a library of proteins are displayed, and passed/panned over a surface saturated with excess copies of a single target molecule. Proteins that remain attached to the targets after washing are amplified and diversified. Over multiple rounds of affinity maturation process, high affinity proteins for the target are enriched.
  • Variations of protein display methods includes mRNA display which is a minimalist protein display method, by which protein identifying nucleic acid sequences can be paired with proteins without necessary modification to the protein sequence.
  • mRNA display mRNA molecules are covalentlv attached to the C-terminus of their encoded proteins.
  • mRNA display works by covalently attaching a puromycin antibiotic molecule to the 3' end of the mRNA, such that during the terminal process of translation, the mRNAs become covalently attached to the C-terminal end of the encoded proteins by ribosomal peptidyl transfer of the puromycin moiety. Affinity maturation is achieved by iterating mRNA display in the desired conditions.
  • cDNA display is an extension of mRNA display techniques wherein a moiety is introduced in the 5' end of a reverse transcription primer.
  • the moiety is generally proximal to the protein such that the moiety can react in a biorthogonal way with the mRN A displayed protein at an unnatural amino acid, or a functional group on the puromycin linker that facilitates covalent attachment of the cDNA to the protein.
  • the mRNA is optionally digested and replaced with DNA to fonn a duplex. Affinity maturation is achieved by iterating cDN A display in the desired conditions.
  • Ribosome display is a display methodology based on known stalling behavior of ribosome-mRNA-protein complexes (RMPCs).
  • RMPCs ribosome-mRNA-protein complexes
  • the non-covalent RPMC is produced from protein coding mRNA transcripts that encode a C-tenninal SecM or equivalent ribosomal stalling sequence, generally preceded by a flexible linker for steric purposes.
  • the RPMC is used directly in panning experiments, and mRNA left behind by the complex is reverse transcribed for PC amplification and diversification of libraries. Affinity maturation is achieved by iterating ribosome display in the desired conditions.
  • Additional protein display techniques include Snap Display, Halo Display, Clip Display, ACP Display and MCP Display where either the C or N terminus of a protein tag reacts with a 5' cDNA or an mRNA terminal moiety to form a covalent bond with the protein tag.
  • These protein tags form robust, physiologically irreversible covalent bonds with their respective ligands upon binding.
  • Exemplary tag and ligand include Halo tag domains that react with Haloalkane ligands, Clip tags that react with benzylcytosine ligands, MCP and ACP tags that react with Acetyl-COA ligand derivatives and Snap tags that react with benzylguanine ligands.
  • the cDNA or mRNA may be attached to the protein tags by in vitro compartmentalized translation reactions, or they may also be produced in a pooled fashion during the production of protein ribosome mRNA complexes wherein the protein tag is coupled to the mRNA or cDNA in Cis.
  • Protein tagged DNA or mRNA complexes can be panned over a substrate coated with binder molecules.
  • the mRNA protein complex can be panned after lysis of the cells, reverse transcribed and/or PCR amplified in an iterative fashion to facilitate affinity maturation and enrichment between the target and binder molecules.
  • Cis display a sequence specific RNA binding protein is added to the C or N terminus of the displayed protein sequence which binds to a biorthogonal sequence motif at the 5' or 3' UTR region in the mRNA encoding the displayed protein.
  • Strict mRNA to protein pairing is accomplished by compartmentalized expression in in vitro compartmentalized translation conditions.
  • cells such as yeast and mammalian cells
  • a centromere containing chromosome can guarantee single copy genetic information, thereby preventing cross coupling of mRNA to other protein species.
  • the mRNA protein complex can be panned after lysis of the cells, reverse transcribed and PCR amplified to facilitate affinity maturation and enrichment.
  • Phage display is a protein display technique that uses bacteriophages to connect proteins with the genetic information that encodes them.
  • the phage is an ml3 phage which contains native proteins in the genome.
  • Exemplary coating proteins on rn !3 phage include pill, pVIII, pVI, pVII and pIX and can be fused to the protein librar ' to be affinity matured.
  • Some bacteriophages used in phage display are m !3 and FD filamentous phage, T4, T7, and ⁇ phage. The DNA packaging behavior of these phages is such that single copy genome packaging can be guaranteed.
  • Phage expressed in E, coli is panned on a surface containing the binder for affinity maturation and enrichment. After washing, the bound phage is allowed to infect a fresh E. coli culture and the process is repeated to achieve affinity maturation and enrichment.
  • Yeast display is a protein display method within a general class of techniques called cell surface display, where the protein of interest is displayed on the cell surface.
  • the protein is displayed by fusion of the protein library to a membrane protein.
  • Classically, cells are panned over a substrate of affinity enrichment binder target. After washing, bound cells are allowed to inoculate fresh media and the process is repeated to achieve affinity maturation and enrichment.
  • the disclosed methods include molecules for affinity maturation that are not solely to proteins and polypeptides.
  • DNA barcoded chemical libraries which permit the production enrichment and diversification of small molecule libraries using hybridization based combinatorial chemistry.
  • Different methods for DNA barcoded small molecule library preparation have been described in Kleiner, R. E., Dumelin, C. E. & Liu, D. R., Small-molecule discovery from DNA-encoded chemical libraries. Chem. Soc. Rev. 40, 5707-17 (201 1) hereby incorporated by reference in its entirety.
  • DNA barcoded library synthesis involves alternating library synthesis steps with enzyme-catalyzed DNA polymerization or ligation of short DNA sequences used to barcode each synthetic step.
  • Library diversity is generated over repeated cycles of division, synthesis, and pooling, a traditional method in combinatorial synthesis.
  • proteins involved in protein-protein interactions are called either bait or prey. Binding of the bait and prey constitutes a protein-protein interaction. These interactions can be determined by a variety of methods known in the art.
  • Co-IP Co-immunoprecipitation
  • Co-IP is a technique used to isolate protein complexes through the use of affinity interactions. Co-IP is typically used in a serial manner to e valuate interactions one protein at a time. It generally requires prior knowledge of likely interacting partners and an antibody to blot for its detection . Co-IP may also be multiplexed using protein and peptide molecule arrays.
  • Mass spectrometiy can be used to determine the identity of co- immunoprecipitated proteins. By targeting a known member with an antibody and utilizing mass spectrometiy it is possible to pull the entire protein complex out of solution and thereby identifying unknown members of the complex. This concept of pulling protein complexes out of solution is sometimes referred to as a "pull-down".
  • the bait protein may be bound by an antibody, or be a constructed protein that can be affinity isolated through a moiety or domain.
  • Yeast Two Hybrid (Y2H) system is another popular method of determining protein- protein interactions that is based on in vivo interactions in yeast.
  • Y2H generally employs a reporter gene whose transcription is governed by a pair of transcriptional cofactors.
  • the two transcriptional cofactors are each fused to a bait and a prey protein, the degree to which the cofactors activate transcription is dependent upon the degree of interaction between the bait and prey proteins.
  • the reporter gene is a fluorescent protein
  • the degree of bait and prey interaction can be quantitated by determining the fluorescent signal via, e.g., flow activated cell sorting and plate based fluorimeter assays of clonal yeast cell populations in wells on multiwell plates.
  • Barcode fusion genetics-Yeast Two-Hybrid is a method by which a full matrix of protein pairs can be screened in a single multiplexed strain pool (Yachie, N. et al., Pooled-matrix protein interaction screens using Barcode Fusion Genetics, Moi. Syst. Biol. 12, 863 (2016) hereby incorporated by reference in its entirety).
  • BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing.
  • BFG-Y2H matrices range in scale from -25 K to 2.5 M protein pairs.
  • BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods.
  • SMS-seq single molecule interaction sequencing
  • DNA barcodes by Halo tag display that is performed in Cis by a stalled ribosome mRNA protein complex. Protein-protein interactions from a panning step are stabilized by cross linking of the protein-protein interface, and the complexes are cast into gels. Polony PCR performed on interacting molecules generates co-localized barcode polonies. Using fluorescent in situ sequencing, the barcode identity co-localization data for each molecule is sufficient to perform library on library screens of interaction in vitro. Furthermore, disassociation constants of molecules could be determined from calculations from the ratio of co-localized and non-co-localized barcode polonies. The methods of the instant disclosure differ from the SMI-seq method in several ways.
  • this disclosure provides methods for the in situ joining and recovery of two coUocalized barcodes to facilitate ex-situ determination of molecule interactions using ex-situ
  • DNA sequencing methods that include, but are not limited to sequencing by synthesis, sequencing by ligation, and sequencing by hybridization instruments. Further, whereas the SMI-seq method permits determination of interactions and affinity maturation, it does not provide methods for the retrieval of the specific subset of binder molecule sequences from the libraiy as herein described by the instant disclosure. This disclosure provides further function beyond the SMI-seq method, including but not limited to: DNA library based sequence retrieval, and interaction profiling by ex-situ DNA sequencing.
  • IDPCR Interaction-dependent PCR
  • IDPCR is a small molecule- protein interaction profiling method based on selectively amplifying DNA sequences encoding ligand-target pairs. IDPCR requires DNA-linked ligands and DNA-linked targets, which, in the publication, were prepared in individual wells by simple nonspecific conjugation. Binding between DNA-linked targets and DNA-linked ligands induces formation of an extendable duplex, where extension links codes that identify the Iigand and target.
  • Orthogonal PCR primers may be random or preassigned.
  • dial-out PCR methods described by Shendure and colleagues (Schwartz, J. J., Lee, C. & Shendure J., Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules, Nat. Methods 9, 913-5 (2012); Klein, J. C. et al., Multiplex pairw!se assembly of array-derived DNA oligonucleotides, Nucleic Acids Res, gkvl l77- (2015). doi: 10.1093/nar/gkvl l 77 hereby incorporated by reference in their entireties), a barcode libraiy is flanked at the 3 ' end by a common PCR sequence that target the library.
  • 5' common adapters are added to facilitate libraiy sequencing. Determination of the assignment of PCR primers to libraiy members is accomplished by next generation sequencing (NGS). Desired sequences can be retrieved independently from the libraiy by PCR employing the determined combination of assigned orthogonal primers to the library member. Alternatively, the orthogonal priming sites may be preassigned to specific members of the libraiy. In the preassigned case, no DNA sequencing of the library-priming site pairs is necessary.
  • NGS next generation sequencing
  • the disclosed methods have the advantage over the conventional methods for determining interactions between proteins by DNA sequencing because the disclosed methods can simultaneously facilitate affinity maturation, interaction profiling and sequence recovery (Table 1).
  • the disclosure provides general methods for multiplexing affinity maturation of target molecule libraries against binder molecule libraries, where the interactions between the target and binder molecules can be determined by determining the barcoded nucleic acid sequences associated with the respective target and binder molecules, and the exact subsets of the molecules in the binder library can be recovered in either pooled or clonal forms by PCR and sequencing.
  • Embodiments of the present invention are directed to nucleic acid sequences have sequences of open reading frame (ORF) encoding the protein molecules and 3' or 5' UTRs.
  • the UTR sequences comprise two or more orthogonal primer binding sites that each hybridizes to an orthogonal primer.
  • orthogonal primer binding site is intended to include, but is not limited to, a nucleic acid sequence located within the UTR sequences of the present invention which hybridizes a complementary orthogonal primer.
  • An “orthogonal primer pair” refers to a set of two primers of identical sequence that bind to both orthogonal primer binding sites within the UTR sequences.
  • Orthogonal primer pairs are designed to be mutually non-hybridizing to other orthogonal primer pairs, to have a low potential to cross-hybridize with one another or to have a low potential to form secondary- structures, and to have similar melting temperatures (Tms) to one another.
  • Orthogonal primer pair design and software useful for designing orthogonal primer pairs is known to a skilled in the art.
  • overlap extension PCR or assembly PCR is used to produce a nucleic acid sequence of interest from, a plurality of nucleic acid sequences.
  • Assembly PCR refers to the synthesis of long, double stranded nucleic acid sequences by performing PCR on a pool of oligonucleotides or nucleic acid sequences having overlapping segments. Assembly PCR is discussed further in Stemmer et al . (1995) Gene 164:49, In certain aspects, PCR assembly is used to assemble single stranded nucleic acid sequences (e.g., ssDNA) into a nucleic acid sequence of interest. In other aspects, PCR assembly is used to assemble double stranded nucleic acid sequences (e.g., dsDNA) into a nucleic acid sequence of interest.
  • “Complementar '” or “substantially complementary” refers to the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid.
  • Complementary nucleotides are, generally, A and T (or A and U), or C and G.
  • Two single- stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from, about 98 to 100%.
  • RNA or DNA strand will hybridize under selective hybridization conditions to its complement.
  • selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90%
  • “Complex” refers to an assemblage or aggregate of molecules in direct or indirect contact with one another.
  • '"contact or more particularly, '"direct contact
  • a complex of molecules in reference to a complex of molecules or in reference to specificity or specific binding, means two or more molecules are close enough so that attractive noncovalent interactions, such as van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules.
  • a complex of molecules is stable in that under assay conditions the complex is ihermodynamically more favorable than a non- aggregated, or non-complexed, state of its component molecules.
  • “complex” refers to a duplex or triplex of polynucleotides or a stable aggregate of two or more proteins. In regard to the latter, a complex can be formed by an antibody specifically binding to its corresponding antigen.
  • 'Duplex refers to at least two oligonucleotides and/or polynucleotides that are fully or partially complementary undergo Watson-Crick type base pairing among all or most of their nucleotides so that a stable complex is formed.
  • annealing and
  • stable duplex means that a duplex structure is not destroyed by a stringent wash, e.g., conditions including temperature of about 5° C. less that the T m of a strand of the duplex and low monovalent salt concentration, e.g., less than 0.2 M, or less than 0.1 M.
  • Perfectly matched in reference to a duplex means that the polynucleotide or oligonucleotide strands making up the duplex form a double stranded structure with one another such that every nucleotide in each strand undergoes Watson-Crick base pairing with a nucleotide in the other strand.
  • duplex comprehends the pairing of nucleoside analogs, such as
  • a "mismatch" in a duplex between two oligonucleotides or polynucleotides means that a pair of nucleotides in the duplex fails to undergo Watson-Crick bonding.
  • Hybridization refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide.
  • Hybridization may also refer to triple -stranded hybridization.
  • the resulting (usually) double-stranded polynucleotide is a “hybrid” or “duplex.”
  • Hybridization conditions will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and even more usually less than about 200 mM.
  • Hybridization temperatures can be as low as 5°C, but are typically greater than 22°C, more typically greater than about 30°C, and often in excess of about 37°C.
  • Hybridizations are usually performed under stringent conditions, i.e., conditions under which a probe will hybridize to its target subsequence.
  • stringent conditions are selected to be about 5°C. lower than the T D , for the specific sequence at s defined ionic strength and pH.
  • Exemplary stringent conditions include salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25° C.
  • Hybridizing specifically to or “specifically hybridizing to” or like expressions refer to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Kit refers to any delivery system for delivering materials or reagents for carrying out a method of the invention.
  • deliver ⁇ ' systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., primers, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another.
  • kit include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials for assays of the invention.
  • Such contents may be delivered to the intended recipient together or separately.
  • a first container may contain an enzyme for use in an assay, while a second container contains primers.
  • Ligation means to form a covalent bond or linkage between the termini of two or more nucleic acids, e.g., oligonucleotides and/or polynucleotides, in a template-driven reaction.
  • the nature of the bond or linkage may vary widely and the ligation may be carried out enzymatically or chemically.
  • ligations are usually carried out enzymatically to form a phosphodiester linkage between a 5 ' carbon of a terminal nucleotide of one oligonucleotide with 3' carbon of another oligonucleotide.
  • a variety of template- driven ligation reactions are described in the following references: Whitely et al., U.S. Pat.
  • “Amplifying” includes the production of copies of a nucleic acid molecule via repeated rounds of primed enzymatic synthesis.
  • "In situ" amplification indicated that the amplification takes place with the template nucleic acid molecule positioned on a support or in a gel, rather than in solution. In situ amplification methods are described in U.S. Pat. No. 6,432,360.
  • “Support” can refer to a matrix upon which molecules of the present invention are placed.
  • the support can be solid or semi-solid or a gel.
  • “Semi-solid” refers to a compressible matrix with both a solid and a liquid component, wherein the liquid occupies pores, spaces or other interstices between the solid matrix elements.
  • Semi-solid supports can be selected from polyacrylamide, cellulose, polyamide (nylon) and crossed linked agarose, dextran and polyethylene glycol.
  • Primer includes an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed.
  • the sequence of nucleotides added during the extension process are determined by the sequence of the template polynucleotide.
  • primers are extended by a D A polymerase. Primers usually have a length in the range of between 3 to 36 nucleotides, also 5 to 24 nucleotides, also from 14 to 36 nucleotides. Primers within the scope of the invention include orthogonal primers, amplification primers, constructions primers and the like. Pairs of primers can flank a sequence of interest or a set of sequences of interest.
  • Primers and probes can be degenerate in sequence. Primers within the scope of the present invention bind adjacent to a target sequence (e.g., an oligonucleotide sequence of an oligonucleotide set or a nucleic acid sequence of interest).
  • a target sequence e.g., an oligonucleotide sequence of an oligonucleotide set or a nucleic acid sequence of interest.
  • temporary orthogonal primers/primer binding sites may be removed using enzymatic cleavage.
  • orthogonal primers/primer binding sites may be designed to include a restriction endonuclease cleavage site.
  • the pool of nucleic acids may be contacted with one or more endonucleases to produce double stranded breaks thereby removing the primers/primer binding sites.
  • the forward and reverse primers may be removed by the same or different restriction endonucleases. Any type of restriction endonuclease may be used to remove the
  • primers/primer binding sites from nucleic acid se uences.
  • restriction endonucleases having specific binding and/or cleavage sites are commercially available, for example, from New England Biolabs (Ipswich, Mass.). In various embodiments, restriction endonucleases that produce 3' overhangs, 5' overhangs or blunt ends may be used.
  • an exonuclease e.g., Reclf, Exonuclease I, Exonuclease T, Si nuclease, Pi nuclease, mung bean nuclease, CEL I nuclease, etc.
  • an orthogonal primer/primer binding site that contains a binding and/or cleavage site for a type IIS restriction endonuclease may be used to remove the temporary orthogonal primer binding site
  • restriction endonuclease recognition site is intended to include, but is not limited to, a particular nucleic acid sequence to which one or more restriction enzymes bind, resulting in cleavage of a DNA molecule either at the restriction endonuclease recognition sequence itself, or at a sequence distal to the restriction
  • Restriction enzymes include, but are not limited to, type I enzymes, type II enzymes, type IIS enzymes, type III enzymes and type IV enzymes.
  • the REBASE database provides a comprehensive database of information about restriction enzymes, DNA methyltransferases and related proteins involved in restriction-modification. It contains both published and unpublished work with information about restriction endonuclease recognition sites and restriction endonuclease cleavage sites, isoschizomers, commercial availability, crystal and sequence data (see Roberts et. al. (2005) Nucl. Acids Res. 33:D230, incorporated herein by reference in its entirety for all purposes).
  • primers of the present invention include one or more restriction endonuclease recognition sites that enable type HS enzymes to cleave the nucleic acid several base pairs 3' to the restriction endonuclease recognition sequence.
  • type IIS refers to a restriction enzyme that cuts at a site remote from its recognition sequence.
  • Type HS enzymes are known to cut at a distances from their recognition sites ranging from 0 to 20 base pairs.
  • Type Hs endonucleases include, for example, enzymes that produce a 3' overhang, such as, for example, Bsr I, Bsm I, BstF5 I, BsrD I, Bts I, Mnl I, BciV I, Hph I, Mbo II, Eci I, Acu I, Bpm I, Mme I, BsaX I, Beg I, Bae I, Bfi I, TspDT I, TspGW I, Taq II, Eco57 1, Eco57M I, Gsu I, Ppi I, and Psr I; enzymes that produce a 5' overhang such as, for example, BsmA I, Pie I, Fau I, Sap I, BspM I, SfaN I, Hga I, Bvb I, Fok I, BceA I, BsmF I, Ksp632 I, Eco31 I, Esp3 I, Aar I; and enzymes that produce a blunt end,
  • Type-IIs endonucleases are commercially available and are well known in the art (New England Bioiabs, Beverly, Mass.). Information about the recognition sites, cut sites and conditions for digestion using type Hs endonucleases may be found, for example, on the Worldwide web at
  • Primers suitable for use in the methods disclosed herein may be designed with the aid of a computer program, such as, for example, DNA Works, Gene20iigo, or using the parameters software described herein.
  • primers are from about 5 to about 500, about 10 to about 100, about 10 to about 50, or about 10 to about 30 nucleotides in length.
  • a set of orthogonal primers or a plurality of sets of orthogonal prim ers are designed so as to have substantially similar melting temperatures to facilitate manipulation of a complex reaction mixture. The melting temperature may be influenced, for example, by- primer length and nucleotide composition.
  • a plurality of sets of orthogonal primers are designed such that each set of orthogonal primers is mutually non-hybridizing with one another. Methods for designing orthogonal primers are described further herein.
  • Solid support refers to a material or group of materials having a rigid or semi-rigid surface or surfaces.
  • at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like.
  • the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations.
  • Microarrays usually comprise at least one planar solid phase support, such as a glass microscope slide. Semisolid supports and gel supports are also useful in the present invention.
  • Specific or “specificity” in reference to the binding of one molecule to another molecule, such as a target to a binder, means the recognition, contact, and formation of a stable complex between the two molecules, together with substantially less recognition, contact, or complex formation of that molecule with other molecules.
  • “specific' 1 in reference to the binding of a first molecule to a second molecule means that to the extent the first molecule recognizes and forms a complex with another molecule in a reaction or sample, it forms the largest number of the complexes with the second molecule. In certain aspects, this largest number is at least fifty percent.
  • molecules involved in a specific binding event have areas on their surfaces or in cavities giving rise to specific recognition between the molecules binding to each other.
  • specific binding examples include antibody-antigen interactions, enzyme-substrate interactions, formation of duplexes or triplexes among polynucleotides and/or oligonucleotides, receptor-ligand interactions, and the like.
  • contact in reference to specificity or specific binding means two molecules are close enough that weak non ⁇ covalent chemical interactions, such as van der Waal forces, hydrogen bonding, base-stacking interactions, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules.
  • the methods disclosed herein comprise amplification of nucleic acids including, for example, oligonucleotides, subassemblies and/or polynucleotide constructs (e.g., nucleic acid sequences of interest). Amplification may be carried out at one or more stages during an assembly scheme and/or may be carried out one or more times at a given stage during assembly. Amplification methods may comprise contacting a nucleic acid with one or more primers that specifically hybridize to the nucleic acid under conditions that facilitate hybridization and chain extension. Exemplary methods for amplifying nucleic acids include the polymerase chain reaction (PCR) (see, e.g., Mullis et al. ( 1986) Cold Spring
  • PCR polymerase chain reaction
  • Polymerase chain reaction refers to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of
  • PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates.
  • the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument.
  • a double stranded target nucleic acid may be denatured at a temperature greater than 90°C, primers annealed at a temperature in the range 50-75° C, and primers extended at a temperature in the range 72-78°C.
  • PCR encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, assembly PCR and the like. Reaction volumes range from a few hundred nanoliters, e.g., 200 mL, to a few hundred microliters, e.g., 200 microliters.
  • Reverse transcription PCR or "RT- PCR,” means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, e.g., Tecott et ai., U.S. Pat. No. 5, 168,038.
  • Real-time PCR means a PCR for which the amount of reaction product, i.e., amplicon, is monitored as the reaction proceeds.
  • Nested PCR means a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon.
  • initial primers in reference to a nested amplification reaction mean the primers used to generate a first amplicon
  • secondary primers mean the one or more primers used to generate a second, or nested, amplicon.
  • Multiplexed PCR means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g. Bernard et ai. (1999) Anal. Biochem.. 273:221-228 (two-color real-time PCR). Usually, distinct sets of primers are employed for each sequence being amplified.
  • Quantitative PCR means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen.
  • methods of determining the sequence of one or more nucleic acid sequences of interest are provided. Determination of the sequence of a nucleic acid sequence of interest can be performed using variety of sequencing methods known in the art including, but not limited to, sequencing by hybridization (SBH), sequencing by ligation (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, T ' aqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing (FISSEQ), FISSEQ beads (U.S. Pat. No. 7,425,431), wobble sequencing (PCT/US05/27695), multiplex sequencing (U.S. Ser. No.
  • allele-specific oligo ligation assays e.g., oligo ligation assay (OLA), single template molecule OLA using a ligated linear probe and a rolling circle amplification (RCA) readout, ligated padlock probes, and/or single template molecule OLA using a ligated circular padlock probe and a rolling circle amplification (RCA) readout
  • OLA oligo ligation assay
  • RCA rolling circle amplification
  • ligated padlock probes single template molecule OLA using a ligated circular padlock probe and a rolling circle amplification (RCA) readout
  • High-throughput sequencing methods e.g., on cyclic array sequencing using platforms such as Roche 454, Illumina Solexa, AB-SOLiD, Helicos, Polonator platforms and the like, can also be utilized. High-throughput sequencing methods are described in U.S. Ser. No.
  • the disclosure provides methods that use mRNA display of both the protein target library and the protein binder library that can facilitate tandem identification of molecular species following their interaction.
  • mRNA display In different versions of mRNA display, several methods are employed to covalently attach the puromycin oligo to the 3' end of the mRNA. In most methods, a 3 ' puromycin modified oligo is attached to a common region of the 3 ' UTR of the mRNA. Attachment can be accomplished by UV crosslinking of a 5' terminal psoralen to the mRNA, and enzymatic ligation of the 5' terminal of the puromycin oligo to the 3 ' OH group of the mRNA sequence.
  • the disclosure provides a novel process of mRNA display which facilitates display of 3' UTR barcodes unique to each protein target and its protein binder respectively, without in libraiy variation of the encoded C-terrninal protein sequence.
  • the 3' UTR barcode region of both target and binder mRNAs serve multiple purposes.
  • the barcode region standardizes the amplicon length and amplification conditions of the identifying barcode sequences, without changing the underlying protein sequence. This greatly simplifies PCR of the barcodes and sequencing processes.
  • placement of the barcode in the 3' UTR as opposed to the 5' UTR of the mR A ensures that the barcodes are in standard proximity during protein-protein interactions regardless of the length of the protein coding sequence involved.
  • the 3' UTR barcode regions in the mRNA of binder and target libraries contain a common TYPE IIS endonuclease recognition sequence tandem to the barcode that facilitates covalent joining of the TV ' PE IIS endonuclease digested target and binder amplicons after PCR amplification.
  • orthogonal priming sequences with common PCR conditions that allow for the enrichment of the individual sequences of interest within a particular library in a pooled or unpooled fashion.
  • the disclosure provides the 3' UTR of the coding sequences having the following common motifs:
  • Linker is a flexible glycine serine polypeptide encoding sequence
  • PolyStop is a repeated sequence of stop codons
  • JCT-Fwd, JCT-Rev, Coml, and Com2 are orthogonal 20 base pair isothermal PCR priming sequences.
  • TypellS is a Type IIS endonuclease recognition site, for example a site recognized by the following enzymes: Hgal, Bbvl, BcoDI, BsniAI, BsmFl, FokL SfaNl, Bbsl, BfuAI, Bsal, BsmBL BspMI, BtgZI, Earl, BspQI, Sapl, or other Type lis DNA endonucleases.
  • Linker is a flexible glycine serine polypeptide encoding sequence
  • PolyStop is a repeated sequence of stop codons
  • JCT-Fwd, JCT-Rev, Coml, and Com2 are orthogonal 20 base pair isothermal PCR priming sequences
  • UMI represents a random 20 base pair unique molecule identifier sequence
  • Barcode represents a set of mutually orthogonal 20 base pair PCR priming sequences
  • TypellS is a Type IIS endonuclease recognition site, for example a site recognized by the following enzymes: Hgal, Bbvl, BcoDI, BsmAI, BsrnFI, Fokl, SfaNI, BbsL BfuAI, Bsa!, BsmBI, BspMI, BtgZI, Earl, BspQI, SapL or other Type lis DNA endonucleases.
  • the mRNA display strategy of the disclosure supports the conjugation of 3' UTR engineered mRNA sequences with the encoded proteins without translation tlirough the UTR region. This is accomplished by depleting all ribosome release factors from the in vitro translation system, and introducing multiple stop codons at the end of the protein coding sequence (applicable to both eukaryotic and prokaryotic in vitro translation systems.)
  • the disclosed system has an equivalent property to conventional mRNA display methods where the ribosome will stall at the end of the protein coding sequence until the puromycin is incorporated, at which point, the protein-mRNA conjugate is free to translocate from the p- site with additional assistance from the ribosome recycling factor (FIGS. 1A and IB).
  • the disclosure provides that when the proteins are of a known set of sequences, the
  • UTR barcode can be preassigned to these proteins, each with a unique barcode.
  • barcodes can be randomly assigned to proteins.
  • the UTR barcodes can be fused with the ORF encoding the proteins by methods known in the art, for example, by Golden Gate insertion, Gibson assembly, or the like.
  • the Barcode-ORF pairs must be sequenced fully to recover a barcode association map.
  • the unique Barcode-ORF pairs must be enriched from the population.
  • the UTR barcoding scheme presupposes a unique assignment of each barcode sequence to each displayed protein.
  • the entire ORFeome library containing plasmid DNAs encoding QRFs of the protein targets and binders can be barcoded with preassigned barcodes in a one pot process provided thai the sequence 3' or 5' to the ORF sequence is constant, the plasmids at the site 3' or 5 'to the ORF will be digested as close as possible to the ORF sequence.
  • the linearized plasmids can be recircularized by Gibson Assembly using a pool of barcoding oligonucleotides described herein and as illustrated in FIG. 2,
  • UTR Barcodes as illustrated in FIG. 2 are contained in the following constructs of the libraries.
  • a pair of target and binder molecules can also be named as a pair of bait and prey.
  • An exemplary Bait has the following motif:
  • Linker is a flexible glycine serine polypeptide encoding sequence
  • PoiyStop is a repeated sequence of stop codons
  • Com2 are orthogonal 20 base pair isothermal PCR priming sequences.
  • Type IIS is a Type IIS endonuclease recognition sequence, for example a sequence recognized by the following enzymes: Hgal, Bbvl, BcoDI, BsmAI, BsmFI, Fokl, SfaNI, Bbsl, BfuAI, Bsal, BsmBI, BspMI, BtgZI, Earl, BspQI, Sapl, or other Type lis DNA endonucleases.
  • ORF is the open reading frame of DN
  • 'ORFeome' A sequence of a set of genes collectively termed 'ORFeome', AttLl and AttL2 are orthogonal Phage Recombinase recognition sequences
  • Linker is a flexible glycine serine polypeptide encoding sequence
  • PolyStop is a repeated sequence of stop codons
  • JCT-Fwd JCT-Rev
  • Com 1 are orthogonal 20 base pair isothermal PCR priming sequences.
  • Type IIS is a Type IIS endonuclease recognition site, for example a site recognized by the following enzymes: Hgal, Bbvl, BcoDI, BsmAI, Bsm.FI, Fokl, SfaNL Bbsl, BfuAI, Bsal, BsmBl, BspMI, BtgZI, Earl, BspQI, Sapl, or other Type IIS DNA endonucleases.
  • Gibson Assembly with barcoding pools permits direct assignment of barcodes to specific ORF sequences.
  • barcoded sequences will be recircularized.
  • These recircularized sequences can be selectively amplified by rolling circle amplification (RCA), inverse PGR, transformation or plasmid purification from E. coli.
  • ORFeomes Methods for cloning ORFeomes are known in the art and have been descirbed in Rajagopaia, S. V et al.
  • the Escherichia coli K-12 ORFeome a resource for comparative molecular microbiology, BMC Genomics 11, 470 (2010) and Brasch, M. A., Hartley, J. L. & Vidal, M., ORFeome cloning and systems biology: standardized mass production of the parts from the parts-l ist, Genome Res, 14, 2001-9 (2004), hereby incorporated by reference in their entireties.
  • ORFeomes that are maintained in Gateway vectors have been described in Hartley, J.
  • Cas9 and in vitro prepared guide RNAs can cleave within 6 base pairs to the ORF sequence.
  • the disclosure provides using Cas9 guide RNA for cloning of barcoded ORFeome libraries. In the Cas9 guide RNA mediated digestion, only 2 amino acids of Oterminai scar sequence is generated.
  • In vitro Cas9 guide RMA digestion of ORFeome plasmids is a more general and robust digestion strategy for pooled barcoding of ORFeomes, which is contemplated by the disclosure.
  • the disclosure provides that UTR barcoding DNA libraries of oligos as large as lxlO 6 sequences and up to iSObp in length can be ordered from commercial source such as from the Agilent Oligo Library Service.
  • the disclosure provides DNA scaffolds which were designed with AttR cloning sites that provide the requisite transcription and translation motifs for prokaryotic and eukaryotic in vitro transcription and translation, as well as fusion with protein immobilization tag either N-terminal or C-terminal to the proteins. These DNA scaffolds facilitate one pot preparation of the entire ORFeomes for general expression and mRNA display (FIG. 3).
  • AttR2 5' ⁇ AttR2, T7 Promoter, IRES, Kozak, protein immobilization tag, Linker, AttRl ⁇ 3'
  • AttR2 and AttRl are orthogonal Phage Recombinase recognition sequences
  • IRES is an internal ribosomal entry sequence
  • Kozak is the Kozak consensus sequence
  • HaloTag is an exemplary protein immobilization tag: Haloalkane dehalogenase mutant from Rhodococus, and Linker is a flexible glycine serine polypeptide encoding sequence. Puromycin Oligo Composition and UTR Design for Attachment
  • the disclosure provides attaching puromycin oligo to a common region (Com) of the 3' UTR.
  • the sequence Com l or Com2 may vary depending on the puromycin oligo conjugation method used. It is desirable to keep the 3' UTR short where possible.
  • a 3' puromycin modified oligo is attached to a common region of the 3' UTR. Attachment process disclosed herein includes UV crosslmking of a 5' terminal psoralen to the mRNA 3' UTR, and enzymatic ligation of the 5' tenninai of the puromycin oligo to the 3' OH terminal of the mRNA 3' UTR sequence.
  • the puromycin oligo DNA sequences for UV crosslmking are as follows:
  • psoralen is the UV active crosslinking motif
  • Coml and Com2 are orthogonal 20 base pair isothermal priming sequences
  • C9 represents a C9 flexible linker sequence
  • Puromycin is the puromycin antibiotic. * indicates reverse compliment sequence of Com.
  • the sequence UAA is appended to the 3' terminal sequence of the UTR barcode to facilitate psoralen crosslinking on hybridization, introducing local adenine niicleobases has been demonstrated to increase the efficiency of psoralen UV crosslinking and has been described in Kurz M et al., Nucleic Acids Res., 2000 Sep 15;28( 18):E83, Psoralen photo- crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA -protein fusions, hereby incorporated by reference in its entirety.
  • the puromycm oligo DNA sequences for Splint ligation are as follows:
  • Coml(l 1-20) and Com2(l 1-20) are the latter 10 base pairs of coml and com2 sequences.
  • Com l and Corn2 are orthogonal 20 base pairs isothermal priming sequences and C9 represents a C9 flexible linker sequence, and Puromycin is the puromycin antibiotic.
  • the splint oligo sequences for Splint ligation are as follows:
  • the coml and com2 in the UTR are truncated 10 base pairs from the 3 ' end. This has the effect that the full Coml and Coni2 sequences are produced upon splint ligation. This design shortens the overall UTR length.
  • the disclosure provides protein binder libraries with sequence diversity up to but not limited to, lxlO 15 can be produced with a variety of methods.
  • Exemplar ⁇ ' Protein binders and scaffolds are described in Table 2.
  • Table 2 A list of protein binders and scaffolds.
  • Alphabodies Triple helix coiled coil
  • the methods for assembly and preparation of fixed length UTR barcoded libraries of DNA sequences are known in the art, which includes overlap extension assembly, although Golden Gate assembly may be preferable. Degenerate fixed length libraries can be barcoded in either the 5 ' UTR or 3 ' UTR.
  • the disclosure provides that a complete rnRNA display ready protein library' including barcodes and UMI sequences can be made in a one pot Golden Gate reaction without subsequent amplification. Diverse libraries can be made and diversified by recombination, error prone PGR, and other methods known to a skilled in the art.
  • the disclosure provides that in the mRNA display process according to certain embodiments, as many as a IxlO 15 unique sequences may be simultaneously displayed against a target. In the initial panning of the library against the targets, the diversity of library sequences greatly exceeds the diversity limits of the orthogonal PGR barcodes which may be as diverse as IxlO 6 sequences. Even in the final panning, the diversity of library sequences may remain as high as 1 xlO 8 sequences.
  • the conditions of panning can be tuned such that the barcode sequence diversity is estimated to exceed the diversity of binder library sequences. This diversity and
  • the disclosure provides two strategies to solve this problem.
  • the UMI sequence By separating the functions of the molecular identification and orthogonal PGR based retrieval from the single barcode sequence, the UMI sequence enables a unique identification of every binder sequence, and the orthogonal PGR priming barcode pool can be allowed to be less diverse than the original binder library, yet still sufficiently diverse to enable clonal retrieval PGR after selection in a small number of steps.
  • the libraries of protein binders with predetermined composition are barcoded and identified in a preassigned manner using sequence homology based methods, and degenerate protein binder libraries are barcoded in a randomly assigned manner that ensures that each library sequence is assigned a unique UMI barcode.
  • NNK codon protein libraries When degenerate NNK codon protein libraries, are assembled, they can contain internal stop codons which prematurely truncate translation of the binding protein.
  • NNK refers to a degenerate codon where N is the nucleobase adenine, thymidine, cytosine or guanine, and K is thymidine or guanine.
  • Premature translation termination may, depending on the mRNA display method, lead to expression of competing non-mR A displayed protein.
  • internal stop codons can generation different length protein binders that are undesirable in downstream applications because proteins of different length may introduce variability in immunoprecipitation and complicate detection and staining properties within the libraiy.
  • the disclosure provides methods to filter out constructs that contain premature stop codons from the naive binder libraiy.
  • the entire libraiy of mRNA display is fused with a C-terminal pulldown tag, such that only the full length protein binders are recovered following affinity purification using the pulldown tag, RT-PCR, and gel purification.
  • the refined library is then subsequently ready to use in the initial panning against the target library and subsequent affinity maturation and evolution steps.
  • the disclosure provides methods to prepare for sequence verification of the barcode- library sequences.
  • a single adapter Tn5 tagmentation reaction is first performed. This transposase mediated reaction will add the Illumina P5 priming sequence randomly within the ORF library coding sequence.
  • a limited cycle PGR is performed, which primes the common sequence with an Illumina P7 sequencing adapter and the tagmentati on site to produce a mappable coding sequence-barcode pair.
  • the paired end sequencing primers P5 and P7 are illustrated as ' Illumina PEF and 'Illumina PE2' respectively (FIG. 4).
  • DNA libraries are prepared at a concentration ready for direct in vitro transcription by T7 RNA Polymerase. Approximately one microgram of library DNA is prepared for transcription. 10s to 100s of micrograms of RNA can be produced from a single in vitro transcription reaction. After transcription, the mRNA is processed to remove template DNA from the reaction. DNAse I treatment is the most common method for removing the template DNA. However, the DNAse must be cleared from the solution by methods such as ethanol precipitation, LiCl precipitation or column based RNA cleanup methods. In the alternative, LiCl precipitation can readily recover pure mRNA without the need for DNAse treatment.
  • the mRN A After cleanup, the mRN A is ready for puromycin conjugation.
  • puromycin oligonucleotides As described herein, there are two major methods that covendedly couple puromycin oligonucleotides to the mRNA, which are Splint ligation and UV psoralen crosslinking. The removal of free unconjugated mRNA is critical to ensure that downstream translation produces only mRNA conjugated protein and not free protein.
  • the mRNA-puromycin conjugate is ready for in vitro translation.
  • a variety of methods are known in the art for puromycin- peptide fusion based in vitro translation and mRNA display. These methods involve stalling mRNA secondary structure, stalling mRNA-cDNA duplex, stalling ribosome at stop codons with release factor (RF) depletion as described elsewhere herein.
  • RF release factor
  • In vitro translation may take place in different lysates dependent on the protein to be displayed and required expression conditions.
  • the release factor (RF) depletion strategy is employed which uses the delta RF " PURE system.
  • a eukaryotic expression system will be used.
  • a variety of commercially available eukaryotic systems are available.
  • the commercially available human HeLa cell lysate was used as the eukaryotic expression system for in vitro translation (IVT). This system is also used for validated microarray production.
  • the eukaryotic IVT systems require RNase and protease inhibitors.
  • the Release Factor Regeneration inhibitor was used.
  • Other eukaryotic IVT systems can also be used which include human HeLa cell lysate, rabbit reticulocyte lysate, wheat germ lysate, and insect cell lysate.
  • the mRNA conjugated to the protein must be passivated to an mRNA-cDNA duplex by reverse transcription. This is to prevent interaction biasing mRNA-mRNA interactions, and also avoids in situ reverse transcription which is relatively inefficient.
  • Reverse transcription with MulV Reverse Transcriptase is performed following desalting. The denaturation, annealing, and extension steps in the reverse transcription are kept at or below 37 degrees Celsius to prevent thermal denaturation of the protein-mRNA conjugated complex.
  • the disclosure provides methods for determining binding between libraries of a vari ety of proteins including membrane proteins.
  • properly folded membrane proteins can be mRNA displayed using the translation protocol described herein by performing the in vitro translation in the presence of empty nanodisc assemblies and in the absence of detergent. Protocols for expressing high level of membrane proteins in nanodiscs in in vitro translation systems are known to a skilled in the art and have been described in
  • nanodiscs are composed of a membrane scaffold protein, and a phospholipid, such as palmitoyl-oleoyl-glycero-phosphocholine (POPC) and Dimyristoyl-glycero- phosphocholine (DMPC) as described in Denisov, I. G., Grinkova, Y. V, Lazarides, A. A.
  • POPC palmitoyl-oleoyl-glycero-phosphocholine
  • DMPC Dimyristoyl-glycero- phosphocholine
  • Nanodiscs can be used in cell-free expression reactions to directly integrate the nascent membrane protein into the nanodisc without any detergent added. The optimal concentration of the nanodisc is dependent on the membrane protein expression rate.
  • the example provides methods to mRNA display membrane proteins. Specifically, ORFs of the membrane proteins are pooled together and engineered to express separately both as N and C-terminal protein tagged fusion proteins.
  • the UTR region of the membrane protein ORFeome pool is the same as the non-membrane protein UTR in the ORFeome Pool .
  • DNA barcoded chemical libraries permit the production, enrichment, and diversification of small molecule libraries using hybridization based combinatorial chemistry in combination with affinity maturation techniques.
  • Different methods for DNA directed small molecule library preparation are known to a skilled in the art and have been described and reviewed in Kleiner, R. E., Dumelin, C. E. & Liu, D. R., Small -molecule discover ' from DNA-encoded chemical libraries, Chem. Soc. Rev. 40, 5707- 17 (201 1) hereby incorporated by reference in its entirety.
  • the example uses the general approach to DNA barcoded library' synthesis which involves alternating library synthesis steps with enzyme-catalyzed DNA polymerization or ligation of short DNA sequences used to barcode each synthetic step of small molecules.
  • Library diversity is generated over repeated cycles of division, synthesis, and pooling, a well- established method in combinatorial synthesis.
  • DNA-barcoded combinatorial libraries have been made containing up to 8xl0 8 individual library members as described in Clark, M. A. et al . Design, synthesis and selection of DNA-encoded small -molecule libraries, Nat. Chem. Biol. 5, 647-54 (2009) hereby incorporated by reference in its entirety.
  • the initial DNA duplex sequence is arbitrary, as is the encoded the length of the 3' overhang, which formed a substrate for subsequent ligation to barcoded tags.
  • the DNA coding sequences were also arbitrary in length and size.
  • the barcodmg tags were short double-stranded DNA sequences consisting of a variable region flanked by constant 3' overhangs. The 3' overhangs were unique to each cycle of synthesis, so that each set of tags could only ligate to the set from the preceding cycle, and not to trancated sequences. Because the sequence of the degrees of freedom in the synthetic scheme, the split pool ligation reaction can proceed such that the composed DNA barcode follows the UTR barcode composition:
  • JCT-Rev, and Com2 are orthogonal 20 base pair isothermal PCR priming sequences
  • 'coding' is the assembled molecule coding sequence
  • Barcode represents a set of mutually orthogonal 20 base pair PGR priming sequences
  • TypelTS is a Type IIS
  • endonuclea endonuclea.se recognition sequence, for example a sequence recognized by the following enzymes: Hgal, Bbvl, BcoDl, BsmAL BsmFI, FokL SfaNl, Bbsl, BfuAl, BsaL BsniBL BspMI, BtgZI, Earl, BspQI, Sapl, or other Type IIS DNA endonucleases.
  • the disclosure provides in an affinity maturation process, the proteins of the target library are attached to a surface.
  • an ideal surface attachment is accomplished using a common feature on the target molecule.
  • the surface area for panning should be scaled according to the product of the target and binder library complexity to adequately sample possible interactions (FIG. 5).
  • the common feature on the target molecule for attachment may use a common protein immobilization tag, including but not limited to a Clip Tag, Snap Tag, MCP Tag, ACP Tag or Halo Tag domains.
  • a common protein immobilization tag including but not limited to a Clip Tag, Snap Tag, MCP Tag, ACP Tag or Halo Tag domains.
  • These protein domains form robust, physiologically irreversible covalent bonds with their respective ligands upon binding.
  • These protein domains can be engineered at either the C-terminal or N-terminal of the target proteins.
  • pools of N and C terminal protein tag and target protein fusions can be screened together.
  • Halo Tag domains react with Haloalkane ligands.
  • Clip Tag reacts with benzylcytosine ligands
  • MCP and ACP tags react with Acetyl -CO A iigand derivatives
  • Snap Tags react with benzylguanine ligands.
  • the panmng substrate is a Sepherose resin having a protein immobilization tag iigand
  • Sepherose45 resin provides high surface area to volume ratio of Haloalkane Irgands.
  • the pooled protein immobilization tagged target library were first loaded onto the resin. Next, the remaining protein
  • immobilization tag ligand surface of the resin was saturate with free protein immobilization tags. Due to the nature of this affinity maturation and evolution scheme, it only needed to mRNA display the protein immobilization tagged target library in the last panning step for interaction mapping.
  • a degenerate binder library of mRN A displayed protein-mRNA conjugates that were passivated were panned through the resin, in the present of competitive negative selection components.
  • the negative selection components included free protein immobilization tags and mRNA-cDNA duplexes. The standard mRNA display evolution scheme as described herein is followed for all panning steps.
  • Bound target binder pairs from the panning were treated with a protein crosslinking reagent to stabilize the protein-protein complexes of the target binder pairs (FIG. 6).
  • the crosslinked protein-protein complexes were cleaved from the resin support.
  • Chemical protein cross-linkers are known to a skilled in the art and have been described in the literature such as Hermanson, G. T., Bioconjugate Technique . Bioconjiigate Techniques (Elsevier, 2013), doi: 10.1016/B978-0-12-382239-0.00006-6 hereby incorporated by reference in its entirety.
  • Table 3 provides a non-limiting exemplar ⁇ ' list of crosslinking targets and reacting groups used for the experiment.
  • Table 3 A list of crosslinking targets and reacting groups.
  • cleavage of the crosslinked protein-protein complexes from the resin support can be accomplished in two general ways, either by cleavage of the target protein from its protein tag, or by cleavage of the tag reacted ligand form the resin support.
  • the former approach cleavage from the protein tag, is limited from two standpoints. First it assumes that the crosslinking treatment does not already crosslink the tag domain to the target protein. Second, in a C-terminal tagged construct, cleavage of the target would leave behind the tag and mRNA barcode on the resin following cleavage, and thus would not allow subsequent barcode -barcode coupling.
  • the later approach of cleavage scheme that targets the ligand-resin interface is an ideal approach for releasing of the crosslinked protein-protein complexes.
  • the later cleavage scheme can be generalized across various crosslinking schemes, and is compatible with both N and C terminal tagged constructs.
  • Exemplary cleavabie bonds and cleavage conditions are known in the art and have been discussed in Leriche, G., Chishoim, L. & Wagner, A., Cleavabie linkers in chemical biology, Bioorganic Med. Chern. 2 ⁇ , 571-582 (2012) hereby incorporated by reference in its entirety.
  • Tables 4 and 5 provide a non-limiting list of exemplary cleavabie bonds and groups.
  • Table 4 A List of Exemplary Protein Tag Qeavable Groups
  • MDA malondialdehyde
  • Oxidizing reagents 1 Vicinal diols, selenium compounds Oxidizing reagents 1 Vicinal diols, selenium compounds.
  • the disclosure provides methods for amplifying mRNA-cDNA duplex conjugated to the crosslinked protein-protein target binder pairs. Generally, it is desirable to amplify both the target and binder barcodes of the mRNA-cDNA duplexes prior to barcode transfer to increase the efficiency of the reaction.
  • two solid phase strategies were applied to amplify DNA in situ.
  • an isothermal or thermal cycled PCR. reaction occurs on the solid phase substrate surface, such as silicon or silica, where the PCR primer oligonucleotides are coupled to the surface of the substrate.
  • an isothermal or thermally cycled PCR reaction occurs in a porous solid phase substrate volume.
  • Exemplar ⁇ ' substrates may include polyacrylamide, polyacryiate, PEG matrix structure, or alternative matrix structures, where the PCR oligonucleotide primers are covalently integrated with the porous substrate.
  • the disclosure provides isothermal DNA amplification methods that include nicked strand displacement PCR, where the DNA polymerase is PhiX29 DNA Polymerase, Bstl DNA Polymerase, or Kappa Hifi DNA polymerase.
  • the disclosure provides thermal cycled DNA amplification methods that include polymerase chain reaction (PCR), where the DNA polymerase is Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Kappa DNA polymerase, Phusion DNA polymerase, Q5 DNA polymerase, or other DNA polymerases known to a skilled in the art.
  • PCR polymerase chain reaction
  • the disclosure provides methods for barcode transfer from polonies that uses recombinase and integrase mediated UTR barcode transfer where the recombinase and integrase recognition sequences are immediately flanking the barcode sequences in the UTR.
  • Recombinases are sequence specific and have been described in Gaj, T., Sirk, S. J. & Barbas, C. F., Expanding the scope of site-specific recombinases for genetic and metabolic engineering, Biotechnol. Bioeng. Ill, 1-15 (2014) hereby incorporated by reference in its entirety. Table 6 lists non-limiting examples of recombinases and target sites.
  • overlap extension PCR amplifies a shared complimentary 7 sequence existing at the 3' terminus of the sense or antisense strand of cDNA in the polonies.
  • DNA polymerase used in the overlap extension PCR is Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Kappa DNA polymerase, Phusion DNA polymerase, Q5 DNA polymerase, or other DNA polymerases.
  • the disclosure provides methods for barcode transfer from polonies that uses Golden Gate Assembly methods.
  • Golden Gate Assembly can be used for either 3' and or 5' UTR barcodes where a type IIS restriction endonuclease digestion of polonies produces ligation compatible ends thai are located immediately adjacent to the target and binder barcodes.
  • Golden Gate Assembly can be used to couple the target and binder barcodes.
  • the type IIS restriction endonuclease includes, for example, Hgal, Bbvl, BcoDI, BsmAI, BsmFI, Fokl, SfaNI, Bbsl, BfuAI, Bsal, BsmBI, BspMI, BtgZI, Earl, BspQI, Sapl, or other Type IIS DNA endonucleases.
  • DNA ligase includes, for example: Taq DNA Ligase, T7 DNA Ligase, T3 DNA ligase, T4 DNA ligase, E, coli DNA ligase, and other DNA ligases.
  • the disclosure provides methods for barcode transfer from polonies that uses Gibson Assembly methods.
  • Gibson Assembly can be used for either 3' and or 5' UTR barcodes where sequences immediately adjacent to the target and binder barcodes in the UTR are homologous ends.
  • Enzymes used with Gibson Assembly is a 5' to 3' exonuclease including, for example, T5 exonuclease, T7 exonuclease, Lambda exonuclease, exonuclease VTII or other 5 " to 3 " exonucleases known to a skilled in the art.
  • DNA polymerase used with Gibson Assembly includes, for example, Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase.
  • DNA ligase used with Gibson Assembly includes, for example, Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, T4 DNA ligase, E. coil DNA Ligase, and other DNA ligases.
  • the disclosure provides methods for barcode transfer from polonies that uses Splint ligation methods.
  • Splint ligation splint oligos containing reverse complimentar - sequences immediately adjacent to both the target and binder barcodes are used to ligate two cDNAs to couple the target and binder barcodes.
  • the li gases used with the Splint ligation are T3 DNA ligase, T7 DNA ligase, E. coli DNA !igase, Taq DNA ligase, or other DNA ligases.
  • the disclosure provides that the most challenging aspect of the library against library screening method is the recover of a clean map of each binder to its target protein.
  • the target-binder complex brings two mRN A-cDNA duplex barcodes together in proximity.
  • Methods for joining single molecule DNA sequences, such as proximity ligation and overlap extension PGR are not robust, and can lead to erroneous DNA coupling in trans.
  • the use of polony PGR of interacting molecules to prepare the colocalized Fisseq Amplicons have been described in Gu, L. el al. Multiplex single-molecule interaction profiling of DNA-barcoded proteins. Nature 515, 554-557 (2014) hereby incorporated by- reference in its entirety.
  • Polony PCR methods as herein disclosed provides an excellent solution for amplification of single molecule sequences, whose subsequent ligation is remarkably robust, and spatially isolated from cross- reactions (FIG. 7).
  • the disclosure provides methods for in gel ligation to couple target binder barcodes that utilize a type IIS restriction endonuclease to digest the polonies that produces complimentary overhangs between the binder and target species for ligation in the gel.
  • covalent joining of colocalized DNA polonies by Golden Gate ligation produces a fixed length tandem barcode sequence that can be amplified for sequencing or clonal isolation of the desired binder (FIG. 8 and FIG. 9).
  • the UTR motifs are designed such that when the barcodes are paired in a single cDNA, they can by sequenced by PCR primers using the universal JCT-fwd and JCT-rev sites immediately adjacent to the barcode and UMI sequences (FIG. 10).
  • the in gel iigated cDNAs can be directly prepared for Illumina sequencing by in gel PCR and electroelution or passive elution of the amplicon DNAs from the gel. The process may be done in multiple PC steps.
  • the protein binder can be sequenced to link the UMI sequence information to the variable protein scaffold sequence.
  • the sequencing involves sequencing from JCT-fwd to the Jet-Rev sequence adjacent the binder coding region (FIG. 10 and FIG. 11).
  • the UTR region flanking the binder sequence is universal such that the whole binder barcode-barcode pairs can be sequenced and recovered.
  • the DNA in the gel can be directly prepared for Illumina sequencing by in gel PCR and electroelution or passive elution of amplicon DNAs from the gel. The process may be done in multiple PCR steps.
  • clonal recovery procedure has two steps. First, the barcode-barcode binder amplicon is generically amplified as a mixed pool. The mixed pool is distributed across microplates for as many target binding binders as desired (one binder to be recovered per well). A subsequent PCR against the target barcode recovers ail the binders for a particular target. Next, a final PCR against the binder barcode can isolate the individual binder species that bind to a particular target. In certain embodiments, the two PCR steps may be unnecessary if all the binder species are uniquely barcoded.
  • a pooled enrichment of binders is sufficient for generating widely applicable reagents.
  • a pool of protoprimer oligos termed OLS (oligo library synthesis) are ordered. This pool of protoprimers is amplified and processed to mature primers.
  • a pool of barcode-barcode-binder amplicons are enriched by PCR and are suitable for downstream modifications for affinity maturation.

Abstract

L'invention porte sur un procédé visant à déterminer la liaison moléculaire entre une banque cible de molécules et une banque de molécules de liaison, et consistant à mettre en contact la banque cible de molécules comprenant une pluralité de conjugués ARNm-protéines avec la banque de molécules de liaison comprenant une pluralité de conjugués ARNm-protéines dans des conditions où une ou plusieurs protéines de la banque cible se lient à une ou plusieurs protéines de la banque de liants, et déterminer la liaison moléculaire des paires de protéines cibles et de liaison en déterminant les séquences des ARNm conjugués aux paires de protéines cibles et de liaison.
PCT/US2017/039862 2016-06-30 2017-06-29 Procédé de détermination de la liaison moléculaire entre des banques de molécules WO2018005720A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3604525A1 (fr) * 2018-08-02 2020-02-05 TU Dresden Procédé permettant de fournir une bibliothèque encodée par adn, bibliothèque encodée par adn et procédé de décodage d'une bibliothèque encodée par adn
CN111965295A (zh) * 2020-07-28 2020-11-20 上海药明康德新药开发有限公司 一种基于液质联用技术验证的dna编码苗头化合物的鉴定方法
CN112813505A (zh) * 2020-12-30 2021-05-18 上海交通大学 一种抗原抗体共展示方法用于膜抗原的抗体文库筛选
US11513126B2 (en) 2017-10-31 2022-11-29 Encodia, Inc. Kits for analysis using nucleic acid encoding and/or label
US11634709B2 (en) 2019-04-30 2023-04-25 Encodia, Inc. Methods for preparing analytes and related kits
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof
US11959922B2 (en) 2016-05-02 2024-04-16 Encodia, Inc. Macromolecule analysis employing nucleic acid encoding

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5942609A (en) * 1998-11-12 1999-08-24 The Porkin-Elmer Corporation Ligation assembly and detection of polynucleotides on solid-support
US20100099103A1 (en) * 2008-09-30 2010-04-22 Abbott Laboratories Antibody Libraries
US20130137605A1 (en) * 2008-09-12 2013-05-30 University Of Washington Sequence tag directed subassembly of short sequencing reads into long sequencing reads
US20150099268A1 (en) * 2008-01-14 2015-04-09 Applied Biosystem, Llc Amplification and detection of ribonucleic acids
WO2015148606A2 (fr) * 2014-03-25 2015-10-01 President And Fellows Of Harvard College Puce à protéines à code-barres pour le profilage d'une interaction monomoléculaire multiplex

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5942609A (en) * 1998-11-12 1999-08-24 The Porkin-Elmer Corporation Ligation assembly and detection of polynucleotides on solid-support
US20150099268A1 (en) * 2008-01-14 2015-04-09 Applied Biosystem, Llc Amplification and detection of ribonucleic acids
US20130137605A1 (en) * 2008-09-12 2013-05-30 University Of Washington Sequence tag directed subassembly of short sequencing reads into long sequencing reads
US20100099103A1 (en) * 2008-09-30 2010-04-22 Abbott Laboratories Antibody Libraries
WO2015148606A2 (fr) * 2014-03-25 2015-10-01 President And Fellows Of Harvard College Puce à protéines à code-barres pour le profilage d'une interaction monomoléculaire multiplex

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ENGLER ET AL.: "Golden gate shuffling: a one-pot DNA shuffling method based on type lis restriction enzymes", PLOS, vol. 4, no. 5, 14 May 2009 (2009-05-14), pages e5553-1 - e5553-9, XP002613222 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11959922B2 (en) 2016-05-02 2024-04-16 Encodia, Inc. Macromolecule analysis employing nucleic acid encoding
US11513126B2 (en) 2017-10-31 2022-11-29 Encodia, Inc. Kits for analysis using nucleic acid encoding and/or label
US11782062B2 (en) 2017-10-31 2023-10-10 Encodia, Inc. Kits for analysis using nucleic acid encoding and/or label
EP3604525A1 (fr) * 2018-08-02 2020-02-05 TU Dresden Procédé permettant de fournir une bibliothèque encodée par adn, bibliothèque encodée par adn et procédé de décodage d'une bibliothèque encodée par adn
JP7397464B2 (ja) 2018-08-02 2023-12-13 テーウー ドレスデン Ｄｎａエンコードライブラリーを提供するための方法、ｄｎａエンコードライブラリー、およびｄｎａエンコードライブラリーをデコードする方法
US11634709B2 (en) 2019-04-30 2023-04-25 Encodia, Inc. Methods for preparing analytes and related kits
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof
CN111965295A (zh) * 2020-07-28 2020-11-20 上海药明康德新药开发有限公司 一种基于液质联用技术验证的dna编码苗头化合物的鉴定方法
CN112813505A (zh) * 2020-12-30 2021-05-18 上海交通大学 一种抗原抗体共展示方法用于膜抗原的抗体文库筛选

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