# US 12,006,497 B2 · Chemical methods for nucleic acid-based data storage > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,006,497 B2 - Document ID: US12006497B2 - Status: Granted - Kind: B2 - Application number: 17/674,170 - Filed: 2022-02-17 - Published: 2023-01-26 - Granted: 2024-06-11 - Earliest claimed: 2018-03-16 - Assignees: Catalog Technologies, Inc. - Applicants: Catalog Technologies, Inc. - Inventors: Devin Leake; Milena Lazova; Sarah Flickinger; Nathaniel Roquet; Hyunjun Park; Swapnil P. Bhatia - Web page: https://patentgrep.com/patent/US12006497B2 ## CPC - C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags} - C12Q 1/68: involving nucleic acids - G06N 3/123: DNA computing - G11C 13/0019: {comprising bio-molecules} - G11C 13/02: using elements whose operation depends upon chemical change {(G11C13/0009 takes precedence)} - B01J 2219/00722: Nucleotides - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G06N: COMPUTING ARRANGEMENTS BASED ON SPECIFIC COMPUTATIONAL MODELS - G11C: STATIC STORES (semiconductor memory devices H10B) - B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS ## Abstract The present disclosure discloses methods and systems for encoding digital information in nucleic acid (e.g., deoxyribonucleic acid) molecules without base-by-base synthesis, by encoding bit-value information in the presence or absence of unique nucleic acid sequences within a pool, comprising specifying each bit location in a bit-stream with a unique nucleic sequence and specifying the bit value at that location by the presence or absence of the corresponding unique nucleic acid sequence in the pool. Also disclosed are chemical methods for generating unique nucleic acid sequences using combinatorial genomic strategies (e.g., assembly of multiple nucleic acid sequences or enzymatic-based editing of nucleic acid sequences). ## Claims ### Claim 1 A method for writing information into a nucleic acid sequence, comprising: (a) determining a string of symbols to represent said information; (b) constructing a plurality of components, wherein each individual component of said plurality of components is a nucleic acid molecule having a nucleic acid sequence; (c) generating at least one sticky end of said individual component of said plurality of components; (d) chemically linking together two or more components of said plurality of components via said at least one sticky end of said individual component of said two or more components, thereby generating one or more identifiers, wherein each identifier of said one or more identifiers comprises two or more components, wherein an individual identifier of said one or more identifiers corresponds to an individual symbol in said string of symbols; and (e) selectively capturing or amplifying an identifier library comprising at least a subset of said one or more identifiers wherein said two or more components are from two or more layers and are assembled in any order, wherein each layer of said two or more layers comprises a distinct set of components, and wherein said individual identifier from said identifier library comprises a component from each layer of said two or more layers. ### Claim 2 (depends on 1) The method of claim 1, wherein each symbol of said string of symbols is one of one or more possible symbol values. ### Claim 3 (depends on 2) The method of claim 2, wherein one symbol value at each position of said string of symbols may be represented by the absence of a distinct identifier in the identifier library. ### Claim 4 (depends on 1) The method of claim 1, wherein (c) comprises using an endonuclease to generate said at least one sticky end of said individual component of said plurality of components. ### Claim 5 (depends on 4) The method of claim 4, wherein said at least one sticky end is at a 5′ end or a 3′ end of said individual component. ### Claim 6 (depends on 1) The method of claim 1, wherein (c) comprises generating two sticky ends of said individual component. ### Claim 7 (depends on 1) The method of claim 1, wherein said at least one sticky end comprises a nucleic acid sequence that is selected from the group consisting of sequences listed in Table 4 or Table 5. ### Claim 8 (depends on 1) The method of claim 1, wherein said plurality of nucleic acid sequences stores metadata of said information or conceals said information. ### Claim 9 (depends on 1) The method of claim 1, wherein two or more identifier libraries are combined and wherein each identifier library of said two or more identifier libraries is tagged with a distinct barcode. ### Claim 10 (depends on 1) The method of claim 1, wherein each individual identifier in said identifier library comprises a distinct barcode. ### Claim 11 (depends on 1) The method of claim 1, wherein chemically linking comprises ligating together two or more components of said plurality of components using a reagent comprising a ligase. ### Claim 12 (depends on 11) The method of claim 11, wherein said ligase is a T4 ligase, a T7 ligase, a T3 ligase, or an E. coli ligase. ### Claim 13 (depends on 11) The method of claim 11, wherein said reagent further comprises an additive. ### Claim 14 (depends on 13) The method of claim 13, wherein said additive comprises polyethylene glycol (PEG). ### Claim 15 (depends on 11) The method of claim 11, further comprising inactivating said ligase using a buffer containing EDTA or guanidine thiocyanate. ### Claim 16 (depends on 11) The method of claim 11, wherein said reagent further comprises glycerol molecules. ### Claim 17 (depends on 1) The method of claim 1, wherein chemically linking in (d) comprises using overlap-extension polymerase chain reaction (PCR). ### Claim 18 (depends on 1) The method of claim 1, further comprising dehydrating said identifier library by dehydrating each individual identifier of at least said subset of said one of more identifiers. ### Claim 19 (depends on 1) The method of claim 1, comprising amplifying said one or more identifiers with PCR or linear amplification. ### Claim 20 (depends on 19) The method of claim 19, wherein said PCR has at least 10 cycles. ### Claim 21 (depends on 19) The method of claim 19, wherein said PCR is an emulsion PCR. ### Claim 22 (depends on 1) The method of claim 1, comprising purifying said one or more identifiers by gel electrophoresis or affinity-tagged probes. ### Claim 23 A method for writing information into a nucleic acid sequence, comprising: (a) determining a string of symbols to represent said information; (b) constructing a plurality of sticky-end components, wherein each individual component of said plurality of components comprises a nucleic acid sequence and at least one sticky end; (c) chemically linking together two or more components of said plurality of components via said at least one sticky end of said individual component of said two or more components, thereby generating one or more identifiers, wherein each identifier of said one or more identifiers comprises two or more components, wherein an individual identifier of said one or more identifiers corresponds to an individual symbol in said string of symbols; and (d) selectively capturing or amplifying an identifier library comprising at least a subset of said one or more identifiers wherein said two or more components are from two or more layers and are assembled in any order, wherein each layer of said two or more layers comprises a distinct set of components, and wherein said individual identifier from said identifier library comprises a component from each layer of said two or more layers. ### Claim 24 (depends on 23) The method of claim 23, wherein (b) comprises annealing two oligonucleotides to construct each individual component such that each individual component has said at least one sticky end.