# US 11,078,480 B2 · High-throughput identification of intermolecular interactions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,480 B2 - Document ID: US11078480B2 - Status: Granted - Kind: B2 - Application number: 16/575,124 - Filed: 2019-09-18 - Published: 2020-03-19 - Granted: 2021-08-03 - Earliest claimed: 2018-09-19 - Assignees: California Institute of Technology - Applicants: California Institute of Technology - Inventors: Mitchell Guttman; Sofia A. Quinodoz; Mario R. Blanco; Devdoot Majumdar - Web page: https://patentgrep.com/patent/US11078480B2 ## CPC - C12N 15/1055: {Protein x Protein interaction, e.g. two hybrid selection} - C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags} - G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites} - G01N 33/58: involving labelled substances (G01N33/53 takes precedence) - G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures} - G01N 2458/10: Oligonucleotides as tagging agents for labelling antibodies - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Methods of detecting an interaction between a macromolecule and an interaction partner are described. Kits are also described. ## Claims ### Claim 1 A method of detecting an interaction between a macromolecule and an interaction partner, the method comprising: obtaining a composition comprising a plurality of macromolecules and a plurality of candidate interaction partners; performing two or more iterations of split-and-pool barcoding, each iteration comprising: (i) randomly distributing the composition into a plurality of partitions in fluid isolation from each other; (ii) in the plurality of partitions, barcoding the plurality of macromolecules and the plurality of candidate interaction partners with a combinatorial barcode unit, wherein within each partition, the plurality of macromolecules and the plurality of candidate interaction partners are barcoded with the same combinatorial barcode unit, wherein the plurality of macromolecules and the plurality of candidate interaction partners of different partitions receive different combinatorial barcode units from each other wherein a macromolecule and a candidate interaction partner that interact will assort together in a partition of the plurality of partitions; and (iii) pooling the barcoded plurality of macromolecules and the barcoded plurality of candidate interaction partners comprising the different combinatorial barcode units from the plurality of partitions into a single composition, wherein, after the two or more iterations of split-and-pool barcoding, each macromolecule of the plurality of macromolecules and each candidate interaction partner of the plurality of candidate interaction partners comprises a combinatorial barcode comprising two or more combinatorial barcode units, each combinatorial barcode unit corresponding to an iteration of split-and-pool-barcoding; and obtaining identifying information from the combinatorial barcodes of the plurality of macromolecules and the plurality of candidate interaction partners, wherein the identifying information permits detecting an interaction between a macromolecule of the plurality of macromolecules and a candidate interaction partner of the plurality of candidate interaction partners through the two or more iterations of split-and-pool barcoding, wherein a probability of the interaction between the macromolecule and the candidate interaction partner as being bona fide is proportional to the number of iterations of split-and-pool barcoding. ### Claim 2 (depends on 1) The method of claim 1, wherein each macromolecule of the plurality of macromolecules is a protein, and wherein each macromolecule of the plurality of macromolecules comprises an identifier barcode comprising a polynucleotide comprising a coding sequence of the respective macromolecule. ### Claim 3 (depends on 2) The method of claim 2, wherein the identifier barcode further comprises a covalent polypeptide tag fused to the polynucleotide, and wherein each macromolecule of the plurality of macromolecules further comprises a counterpart polypeptide covalently bound to the polynucleotide. ### Claim 4 (depends on 3) The method of claim 3, wherein the counterpart polypeptide is disposed at an N-terminal region of the protein. ### Claim 5 (depends on 4) The method of claim 4, wherein each macromolecule of the plurality of macromolecules comprising the identifier barcode is made according to a method comprising: fusing the covalent polypeptide tag to the polynucleotide; translating the polynucleotide in vitro, thereby producing the macromolecule comprising the counterpart polypeptide disposed at an N-terminal portion of the macromolecule; and covalently binding the polypeptide tag to the counterpart polypeptide, thereby making the macromolecule comprising the identifier barcode. ### Claim 6 (depends on 3) The method of claim 3, wherein the covalent polypeptide tag and counterpart polypeptide comprise a split CnaB protein; or a Spytag and SpyCatcher; or Isopeptag and pilin-C; or SnoopTag and SnoopCatcher; or DogTag and SnoopTagJr; or SdyTag and SdyCatcher. ### Claim 7 (depends on 3) The method of claim 3, wherein the covalent polypeptide tag is fused to the polynucleotide via a HUH protein, SMCC linkage, or RepB replicase. ### Claim 8 (depends on 2) The method of claim 2, wherein the identifier barcode further comprises a random oligonucleotide barcode of at least 5 nucleotides. ### Claim 9 (depends on 2) The method of claim 2, wherein the identifier barcode further comprises a terminal single-stranded handle sequence, and wherein each combinatorial barcode unit comprises a terminal single-stranded complement of the terminal handle sequence, wherein said barcoding comprises: permitting the terminal single-stranded handle sequences to anneal to the terminal single-stranded complement of a combinatorial barcode unit; and ligating the terminal handle sequences to the terminal single-stranded complement of the combinatorial barcode unit. ### Claim 10 (depends on 1) The method of claim 1, wherein the combinatorial barcode units comprise oligonucleotides, wherein different sequences of the oligonucleotides differentiate different combinatorial barcode units from each other; or wherein the combinatorial barcode units comprise detectable moieties, wherein different detectable moieties differentiate different combinatorial barcode units from each other. ### Claim 11 (depends on 2) The method of claim 2, further comprising sequencing the coding sequence of the macromolecule. ### Claim 12 (depends on 1) The method of claim 1, wherein the plurality of macromolecules are of a library of in vitro translated polypeptides. ### Claim 13 (depends on 1) The method of claim 1, wherein the plurality of macromolecules are selected from the group consisting of proteins, RNA binding proteins, antigen binding proteins, antibodies or fragments thereof, scFvs, nanobodies, and aptamers, or a combination of two or more of any of the listed items. ### Claim 14 (depends on 1) The method of claim 1, wherein the candidate interaction partners are selected from the group consisting of: proteins, peptides, RNAs, DNAs, and small molecules, or a combination of two or more of any of the listed items. ### Claim 15 (depends on 1) The method of claim 1, wherein the composition comprises at least 103 different macromolecules and at least 103 different candidate interaction partners. ### Claim 16 (depends on 1) The method of claim 1, further comprising detecting the interaction between the macromolecule and the candidate interaction partner, detecting an interaction between the macromolecule and a second candidate interaction partner, and detecting a lack of an interaction between the macromolecule, the candidate interaction partner, and the second candidate interaction partner, thereby indicating that the second candidate interaction partner has a probability of interfering with the interaction between the macromolecule and the candidate interaction partner. ### Claim 17 (depends on 1) The method of claim 1, further comprising detecting the interaction between the macromolecule and the candidate interaction partner, detecting an interaction between a second macromolecule and the candidate interaction partner, and detecting a lack of an interaction between the macromolecule, the second macromolecule, and the candidate interaction partner, thereby indicating that the second macromolecule has a probability of interfering with the interaction between the macromolecule and the candidate interaction partner. ### Claim 18 (depends on 1) The method of claim 1, comprising at least 3 iterations of split-and-pool barcoding.