Chemical methods for nucleic acid-based data storage
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).
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 the 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,
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.
2. The method of claim 1 , wherein each symbol in said string of symbols is one of two possible symbol values.
3. The method of claim 2 , wherein one symbol value at each position of said string of symbols is represented by the absence of a distinct identifier in the identifier library.
4. The method of claim 1 , wherein said two or more components are assembled in a fixed order.
5. The method of claim 1 , wherein said two or more components are assembled with one or more partitioning components disposed between two components from different layers of said two or more layers.
6. 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.
7. The method of claim 6 , wherein said at least one sticky end is at a 5' end or a 3' end of said individual component.
8. The method of claim 1 , wherein (c) comprises generating two sticky ends of said individual component.
9. The method of claim 1 , wherein said at least one sticky end comprises the nucleic acid sequence that is selected from the group consisting of sequences listed in Table 4 or Table 5.
10. The method of claim 1 , wherein said plurality of nucleic acid sequences stores metadata of said information or conceals said information.
11. The method of claim 1 , further comprising:
combining two or more identifier libraries; and
tagging each identifier library of said two or more identifier libraries with a distinct barcode.
12. The method of claim 1 , wherein each individual identifier in said identifier library comprises a distinct barcode.
13. The method of claim 12 , wherein said distinct barcode of each individual identifier has a minimum hamming distance from the distinct barcode of other individual identifiers.
14. 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.
15. The method of claim 14 , wherein said ligase is a T4 ligase, a T7 ligase, a T3 ligase, Taq ligase, Chlorella virus ligase, Thermococcus sp. strain 9° N ligase, or an E. coli ligase.
16. The method of claim 14 , wherein said reagent further comprises an additive.
17. The method of claim 16 , wherein said additive crowds said ligase.
18. The method of claim 16 , wherein said additive comprises polyethylene glycol (PEG), dimethyl sulfoxide (DMSO), 1,2-Propanediol (1,2-Prd) glycerol, or polysorbate 20.
19. The method of claim 14 , further comprising inactivating said ligase using a buffer containing EDTA or guanidine thiocyanate.
20. The method of claim 14 , wherein said reagent further comprises glycerol molecules.
21. The method of claim 1 , wherein chemically linking in (d) comprises using overlap-extension polymerase chain reaction (PCR).
22. The method of claim 1 , further comprising dehydrating said identifier library by dehydrating each individual identifier of at least said subset of said one or more identifiers.
23. The method of claim 1 , further comprising amplifying said one or more identifiers with PCR or linear amplification.
24. The method of claim 23 , wherein said PCR has at least 10 cycles.
25. The method of claim 23 , wherein said PCR is an emulsion PCR.
26. The method of claim 1 , further comprising purifying said one or more identifiers by gel electrophoresis or affinity-tagged probes.
27. 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,
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.
28. The method of claim 27 , wherein (b) comprises annealing two oligonucleotides to construct each individual component such that each individual component has said at least one sticky end.