Non-amplifiable polynucleotides for encoding information
Polynucleotides used for encoding information are synthesized with universal base analogs that participate in pi-stacking interactions but do not form Watson-Crick hydrogen bonds with other bases. The universal base analogs may have pyrrole-based bases such as 5-nitroindole (5NI). Inclusion of the universal base analogs in the polynucleotides prevents polymerase-based amplification such as PCR. However, the non-amplifiable polynucleotides are able to hybridize to complementary strands and the sequences may be read by nanopore sequencing. The polynucleotides may be used as molecular taggants to label items for the prevention of forgery. The ability of polynucleotides collected from an item to hybridize with known sequences can be used to establish authenticity of an item. Alternatively, the polynucleotides may be used to encode digital data in a read-only molecule that cannot be readily copied. The digital data can be retrieved from the polynucleotides by nanopore sequencing and decoding of the nucleotide sequence data.
1 . A method comprising:
applying a polynucleotide taggant to an item, the polynucleotide taggant comprising:
a payload region encoding a unique identifier that is associated with the item; and
at least two universal base analogs that form pi-stacking as part of a double-stranded polynucleotide but do not form Watson-Crick hydrogen bonds with complementary bases, wherein an arrangement of the universal base analogs is such that the polynucleotide taggant is incompatible with polymerase-based amplification;
collecting the polynucleotide taggant from the item; and
determining that the polynucleotide taggant encodes the unique identifier without amplification or copying of the polynucleotide taggant.
2 . The method of claim 1 , wherein determining that the polynucleotide taggant encodes the unique identifier comprises sequencing the polynucleotide taggant by nanopore sequencing.
3 . The method of claim 1 , wherein determining that the polynucleotide taggant encodes the unique identifier comprises detecting hybridization of the polynucleotide taggant to an other polynucleotide.
4 . The method of claim 1 , wherein the universal base analogs are 5-nitroindole (5NI).
5 . The method of claim 1 , wherein the polynucleotide taggant comprises at least one universal base analog on each side of the payload region and no universal base analogs interspersed within the payload region.
6 . The method of claim 1 , wherein the universal base analogs are interspersed within the payload region.
7 . The method of claim 1 , further comprising receiving the polynucleotide taggant from a supplier and creating an electronic record associating the unique identifier with a description of the item.
8 . The method of claim 1 , wherein the unique identifier encodes digital data that is a string of bits.
9 . The method of claim 1 , wherein the universal base analogs are pyrrole-based bases.
10 . The method of claim 1 , wherein applying the polynucleotide taggant to the item comprises applying the polynucleotide taggant in proximity to a visible taggant that is also placed on the item.
11 . The method of claim 1 , wherein determining that the polynucleotide taggant encodes the unique identifier comprises:
sequencing the polynucleotide taggant to obtain a tag sequence;
comparing the tag sequence to a reference sequence; and
identifying a match when a sequence identity between the tag sequence and the reference sequence is at least a threshold level of similarity.
12 . The method of claim 1 , wherein the polynucleotide taggant comprises a mixture of two or more different non-amplifiable polynucleotides and determining that the polynucleotide taggant encodes the unique identifier comprises evaluating a detected pattern of hybridization.
13 . The method of claim 12 , wherein the detected pattern of hybridization comprises a spatial pattern of fluorescence on a substrate comprising spatially localized probes.
14 . A system comprising:
means for applying a polynucleotide taggant to an item, the polynucleotide taggant comprising:
a payload region encoding a unique identifier that is associated with the item; and
at least two universal base analogs that form pi-stacking as part of a double-stranded polynucleotide but do not form Watson-Crick hydrogen bonds with complementary bases, wherein an arrangement of the universal base analogs is such that the polynucleotide taggant is incompatible with polymerase-based amplification;
means for collecting the polynucleotide taggant from the item; and
means for determining that the polynucleotide taggant encodes the unique identifier without amplification or copying of the polynucleotide taggant.
15 . The system of claim 14 , wherein the means for applying the polynucleotide taggant to the item comprises means for coating, mixing, spraying, or encapsulating the polynucleotide taggant in or on the item.
16 . The system of claim 14 , wherein the means for collecting the polynucleotide taggant from the item comprises a swab, rinsing apparatus, or DNA collection and purification kits.
17 . The system of claim 14 , wherein the means for determining that the polynucleotide taggant encodes the unique identifier comprises sequencing the polynucleotide taggant by nanopore sequencing.
18 . The system of claim 14 , wherein the means for determining that the polynucleotide taggant encodes the unique identifier comprises detecting hybridization of the polynucleotide taggant to an other polynucleotide.
19 . The system of claim 14 , wherein the means for determining that the polynucleotide taggant encodes the unique identifier comprises:
sequencing the polynucleotide taggant to obtain a tag sequence;
comparing the tag sequence to a reference sequence; and
identifying a match when a sequence identity between the tag sequence and the reference sequence is at least a threshold level of similarity.
20 . The system of claim 14 , wherein the polynucleotide taggant comprises a mixture of two or more different non-amplifiable polynucleotides and the means for determining that the polynucleotide taggant encodes the unique identifier comprises evaluating a detected pattern of hybridization.