Counterfeit protection using DNA

Abstract

There is a demand for reliable, durable, and accurate methods of authentication in markets of specialty goods, e.g., luxury items and / or security-sensitive products. Currents methods of object authentication, including the incorporation of extrinsic and / or intrinsic markers, remain amenable to counterfeiting. This disclosure provides methods of object authentication and counterfeit protection using DNA sequences, including having multiple data cassettes assigned to a given bit code, and writing data using a mixture of the multiple cassettes in predetermined proportions, thereby providing validation and authenticity of the data.

Description

In markets for specialty goods (such as luxury goods and / or security-sensitive products), there is a demand for reliable, durable, and accurate authentication methods. Existing item authentication methods (including the introduction of exogenous and / or endogenous markers) remain susceptible to counterfeiting. This disclosure provides a method for item authentication and anti-counterfeiting protection using DNA sequences, the method comprising assigning multiple data boxes to a given bit code and writing data using a mixture of the multiple data boxes in a predetermined proportion, thereby achieving data verification and authenticity determination. Abstract

Claims

CLAIMSWhat is claimed is:

1. A population of deoxyribonucleic acid (DNA) sequences encoding data useful in the authentication of objects and for protection against counterfeiting (e.g., selected from DNA 1, et seq. and / or DNA 2, et seq.), comprising nucleic acid data packets (“nackets”), wherein each nacket is encoded by a plurality of DNA molecules encoding the same data, wherein the sequences of the DNA molecules are heterogeneous.

2. The population of DNA sequences of claim 1, wherein the DNA sequences arc prepared using heterologous cassette data writing, wherein two or more cassette sequences are provided for a single bit or combination of bits in a machine-readable code, such that all or nearly all of the DNA molecules in the nacket encode the same data, but the sequences of the individual molecules exhibit extremely high variation, wherein the nackets comprise a plurality of heterologous cassettes.

3. The population of DNA sequences of claim 1 or 2, wherein the data is in n-bit code wherein n is greater than 1, e.g., binary or ternary code.

4. The population of DNA sequences of any foregoing claim, wherein the DNA sequences are prepared from heterologous cassettes encoding the same bit or bits of data, wherein the percent abundance of the different cassette variants used in writing the DNA provides a unique and distinguishable feature of the DNA.

5. The population of DNA sequences of any foregoing claim, wherein the data encoded in the DNA is a nonfungible token (NFT).

6. The population of DNA sequences of any foregoing claim, wherein the one or more DNA sequences and / or cassettes contain one or more topoisomerase recognition sequences,e.g., wherein the topoisomerase recognition sequence is 5’-CCCTT-3’, 5’-TCCTT-3’, 5’- CCCTG-3’, or 5’-TGACT-3’.

7. The population of DNA sequences of any foregoing claim, wherein the DNA comprises cassettes, wherein each cassette comprises (i) an information domain having sequence which corresponds to one or more bits in a machine-readable code, and (ii) a topoisomerase recognition sequence, wherein the cassette is 18-25 nucleotides in length.

8. The population of DNA sequences of any foregoing claim, wherein the DNA is incorporated into or associated with goods for purposes of identifying and authenticating the goods.

9. The population of DNA sequences of any foregoing claim, wherein the DNA is adsorbed onto, incorporated into, or encapsulated by silica beads or particles.

10. A method of object authentication (e.g., according to any of Method 1, et seq., supra), comprising: i. synthesizing a population of DNA sequences, e.g., according to claim 1 , comprising nucleic acid data packets (“nackcts”), wherein each nackct contains a plurality of DNA molecules encoding the same data, wherein the sequences of the DNA molecules are heterogeneous; ii. incorporating said DNA sequences into or onto an object; iii. extracting said DNA sequences from the object; and iv. analyzing the extracted DNA sequences; v. optionally, comparing the analyzed DNA sequences to a database of DNA sequences; vi. optionally, confirming object authenticity.

11. The method of claim 10 wherein the DNA sequences are synthesized by sequential addition of DNA cassettes to DNA receptor strands, wherein in each sequential addition step the cassettes comprise a heterologous population of synonymous cassettes, such that thecassettes have at least two different sequences encoding the same data in a machine- readable code (c.g., binary or ternary code).

12. The method of claim 10 or 11, wherein the cassettes are conjugated together using a ligase enzyme.

13. The method of claim 10 or 11, wherein the cassettes are conjugated together using a topoisomerase enzyme.

14. The method of any of claims 10 to 13, wherein the DNA sequences comprise DNA sequences synthesized using a transferase-based synthesis and data encoding.

15. The method of any previous claim wherein the nackets are synthesized by sequential addition of cassettes to DNA receptor strands using an inkjet printing head (e.g., a piezoelectric print head), wherein each cassette comprises multiple nucleotides, wherein in each sequential addition step the cassettes comprise a heterologous population of cassettes of at least two different sequences encoding the same data in a machine-readable code (e.g., binary or ternary code), and wherein the cassettes are dispensed by an inkjet writing print head on at least one writing spot on a wafer array, the head or nozzle writing the same code to a plurality of polymer memory strands dispensed on the at least one spot, e.g., comprising the following steps: a) loading the desired spot to be written with a starter polymer or DNA attached at one end to the desired spot; b) washing the surface of the spot; c) positioning an inkjet nozzle having a heterologous population of cassettes wherein the population comprises cassettes having at least two different sequences, but all encoding the same information in one or more bits (e.g., 1 or 0, or 00, 01, 10, 11, etc. in binary code) over the desired spot to be written corresponding to the unique code; d) causing the inkjet nozzle to release a droplet comprising the heterologous population of cassettes onto the spot, thereby writing a bit or portion of the unique code to the DNA or polymer memory strings (or strands) associated with the spot; ande) washing the surface of the spot; optionally further comprising steps f) - i): f) causing the inkjet nozzle to release a droplet of deblock / adapter reagent onto the spot; g) washing the surface of the spot; h) repeating steps (c) through (g) until the unique code has been written in the memory string at the spot; and i) removing the memory strings from the spot and flowing the memory strings from the spot into a collection or storage container for later incorporation into or onto an object.

16. The method of claim 15, wherein the cassettes are added by topoisomerase mediated ligation; for example, by:(i) reacting double- stranded acceptor DNA strands with topoisomerases charged with double-stranded DNA cassettes from the heterologous population of cassettes covalently bound to the topoisomerases, wherein a strand of the acceptor DNA has a 5’ overhang, wherein each cassette comprises an informational sequence, a topoisomerase recognition sequence, and 5’ overhangs on both strands, wherein the 5’ overhang of the strand of the oligomer that does not bear the topoisomerase (“bottom strand”) is complementary to the 5' overhang of the acceptor DNA but is not complementary to the 5’ overhang of the strand bearing the topoisomerase (“top strand”) of the cassette, wherein the 5’ end of the strand bearing the topoisomerase (“top strand”) of the cassette and 5’ end of the acceptor DNA are not protected, e.g., not phosphorylated (i.e., 5’-OH), and wherein the topoisomerase charged with a double- stranded DNA cassette is delivered to the location of the acceptor strand by a piczo-electric inkjet nozzle;(ii) reacting the acceptor DNA thus extended in step (i) with a topoisomerase charged with a further double-stranded DNA cassette,wherein the further cassette comprises an informational sequence that is the same as or is different from any informational sequence in the cassette of step (i), a topoisomerase recognition sequence, and 5’ overhangs on both strands, wherein the 5’ overhang of the strand of the further cassette not bearing the topoisomerase (“bottom strand”) is complementary to the 5' overhang of the extended acceptor DNA but is not complementary to the 5’ overhang of the strand of the further cassette bearing the topoisomerase (“top strand”), and wherein the 5’end of the strand bearing the topoisomerase (“top strand”) of the further cassette is not protected, e.g., not phosphorylated (i.e., 5’-OH); and(iii) repeating steps (i) and (ii) until the desired nucleotide sequence is obtained; wherein there is optionally a washing step after step (i) and / or after step (ii); and optionally, wherein the desired nucleotide sequence thus obtained is further reacted with a terminal sequence comprising one or more replication primers, such as one or more PCR primer sequences.

17. A method (e.g., according to any of Method 2, et seq., supra.) of any preceding claim wherein the nackets or cassettes used to make the nackets comprise a desired code, e.g., a ternary code, using a DNA or polymer strand or memory string, wherein the data is encoded in a series of transitions between non-identical nucleotides, with one bit for each such transition, comprising: i. providing a reaction mixture comprising one or more transferase enzyme, e.g., terminal deoxynucleotidyl transferase (TdT) and one or more dNTP degrading enzyme, e.g., apyrase; ii adding to the reaction mixture deoxyribonucleotide triphosphates (dNTPs), e.g., selected from dATP, dCTP, dGTP, and dTTP; iii. waiting until the dNTPs of step (ii) are added or degraded; iv. repeating steps (ii) and (iii) until the desired bit sequence is reached, wherein nonidentical dNTP species are used in any two consecutive additions thereby providing a population of DNA molecules encoding the desired data string.

18. A method of object authentication (e.g., according to any of Method 3, et seq., supra), comprising: i. synthesizing one or more DNA sequences, e.g., according to claim 1, comprising nucleic acid data packets (“nackets”), wherein each nacket contains a plurality of DNA molecules encoding the same data, wherein the sequences of the DNA molecules are synthesized using one or more transferase enzymes, e.g., according to any of DNA 2, et seq., supra,' ii. incorporating said one or more DNA sequences into or onto an object; iii. extracting said one or more DNA sequences from the object; and iv. analyzing the extracted one or more DNA sequences; v. optionally, comparing the analyzed one or more DNA sequences to a database of DNA sequences; vi. optionally, confirming object authenticity.

19. A method for writing an attack resistant digital code using DNA, comprising: i. receiving a desired digital code to be written, the desired code being grouped into four two-bit binary codes to be written (e.g., 00, 01, 10, 11); ii. providing four predetermined mixtures of a predetermined number of unique DNA cassette strings, each mixture corresponding to a different predetermined two-bit binary code value, each mixture having a predetermined proportion of the unique DNA cassettes within the mixture, and the unique DNA cassette strings of each mixture being different from the DNA cassette strings in the other mixtures; iii. depositing a droplet of the mixture associated with a given two-bit binary code to be written onto a substrate to add a DNA cassette string to an encoded DNA string being written, the droplet comprising the predetermined mixture of the unique cassettes associated with the given two-bit binary code; and iv. repeating the depositing until the desired code is written onto the encoded DNA string.

20. The method of claim 19, further comprising, after the desired code is written, adding an end cap to the encoded DNA string.21 . The method of claim 20, wherein the end cap contains information about the desired digital code or how to read the code.

22. The method of any of claims 19 to 21, wherein the substrate has an acceptor DNA strand having one end attached to the substrate and an opposite end being available to attach to one of the unique DNA cassettes to be added.

23. The method of any of claims 19 to 22, wherein the predetermined number of unique DNA cassettes for one of the mixtures is different from at least one other of the mixtures.

24. The method of any of claims 19 to 23, wherein the desired digital code is encoded in an NFT with authentication data and stored on a blockchain.

25. The method of any of claims 19 to 24, wherein the encoded DNA string is embedded in a physical object to be authenticated.

26. A method for writing an attack resistant digital code using DNA, comprising: i. receiving a desired digital code to be written, the desired code being grouped into a plurality of n-bit binary codes to be written, where n is greater than 1; ii. providing at least two predetermined mixtures of a predetermined number of unique DNA cassette strings, each mixture corresponding to a different predetermined zz-b i t binary code value, each mixture having a predetermined proportion of the unique DNA cassettes within the mixture, and the unique DNA cassette strings of each mixture being different from the DNA cassette strings in the other mixtures; iii. depositing a droplet of the mixture associated with a given zz-bit binary code to be written onto a substrate to add a DNA cassette string to an encoded DNA string being written, the droplet comprising the predetermined mixture of the unique cassettes associated with the given zz-bit binary code; and iv. repeating the depositing until the desired code is written onto the encoded DNA string.

27. The method of claim 26, further comprising, after the desired code is written, adding an end cap to the encoded DNA string.

28. The method of claim 27, wherein the end cap contains information about the desired digital code or how to read the code.

29. The method of any of claims 26 to 28, wherein the substrate has an acceptor DNA strand having one end attached to the substrate and an opposite end being available to attach to one of the unique DNA cassettes to be added.

30. The method of any of claims 26 to 29, wherein the predetermined number of unique DNA cassettes for one of the mixtures is different from at least one other of the mixtures.

31. The method of any of claims 26 to 30, wherein the desired digital code is encoded in an NFT with authentication data and stored on a blockchain.

32. The method of any of claims 26 to 31, wherein the encoded DNA string is embedded in a physical object to be authenticated.

33. A method for writing an attack resistant digital code using DNA, comprising: i. receiving a desired digital code to be written, the desired digital code being grouped into four two-bit binary codes to be written (e.g., 00, 01, 10, 11); ii. providing four sets of unique DNA cassette strings, each set comprising a predetermined number of unique DNA cassettes and each set corresponding to a different predetermined two-bit binary code value, such that each set of unique cassettes corresponding to different two-bit binary code and each set of unique cassette strings being different from the other DNA cassette strings; iii. randomly selecting one of the unique cassettes corresponding to a given two-bit binary code to be written, as a selected unique cassette;iv. depositing a droplet of the selected unique cassette associated with the given two-bit binary code to be written onto a substrate to add the selected unique cassette to an encoded DNA string being written; v. repeating the selecting and depositing until the desired code is written onto the encoded DNA string on a given writing spot on the substrate; and vi. counting the number of times each unique cassette is used for each two-bit binary code written.

34. A method for writing an attack resistant digital code using DNA, comprising: i. receiving a desired digital code to be written, the desired digital code being grouped into a plurality of zz-bit binary codes to be written, where n is greater than 1; ii. providing at least two sets of unique DNA cassette strings, each set comprising a predetermined number of unique DNA cassettes and each set corresponding to a different predetermined n-bit binary code value, such that each set of unique cassettes corresponding to different n-bit binary code and each set of unique cassette strings being different from the other DNA cassette strings; iii. randomly selecting one of the unique cassettes corresponding to a given n-bit binary code to be written, as a selected unique cassette; iv. depositing a droplet of the selected unique cassette associated with the given zz-bit binary code to be written onto a substrate to add the selected unique cassette to an encoded DNA string being written; v. repeating the selecting and depositing until the desired code is written onto the encoded DNA string on a given writing spot on the substrate; and vi. counting the number of times each unique cassette is used for each zz-bit binary code written.

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