Use of DNA origami nanostructures for molecular information based data storage systems

Patent metadata

Field	Value
Publication number	US-12441996-B2
Application number	US-202418972564-A
Country	US
Kind code	B2
Filing date	Dec 6, 2024
Priority date	Dec 8, 2023
Publication date	Oct 14, 2025
Grant date	Oct 14, 2025

Abstract

The present disclosure is directed to compositions and methods that use the principles of DNA origami to package and archive data stored in multiple indexed DNA oligonucleotides. These structures allow for selective physical data access and retrieval from a molecular pool of DNA origami (DNAO) nanostructures comprising the data bearing oligonucleotides.

First claim

The invention claimed is:

1. A library comprising a plurality of origami folded DNA data storage files (DNAFiles), each of said DNAFiles comprising
a single stranded DNA scaffold; and
a plurality of single stranded DNA staple oligonucleotides that each bind through complementary base pairing with two non-contiguous nucleic acid sequences of the DNA scaffold, wherein said staple oligonucleotides cause the DNA scaffold to fold into a two or three dimensional shape having a first surface;
a plurality of data oligonucleotides, said data oligonucleotides comprising a sequence complementary to a nucleic acid sequence of said single stranded DNA scaffold, a nucleic acid sequence that encodes digital information, a first primer binding sequence and a second primer binding sequence, wherein the first primer binding sequence is 5′ to the digital information encoding nucleic acid sequence, and the second primer binding sequence is 3′ to the digital information encoding nucleic acid sequence, wherein said plurality of data oligonucleotides are localized to said first surface, wherein the individual DNAFiles differ from one another based on the nucleic acid sequences of the plurality of data oligonucleotides bound to the DNA scaffold of each DNAFile.

2. The library of claim 1 wherein said staple oligonucleotides cause the DNA scaffold to fold into a multi-layered sheet conformation having a top surface and a bottom surface wherein said plurality of data oligonucleotides are only linked to, and project away from, the top surface.

3. The library of claim 2 wherein each DNAFile has a bilayer sheet conformation comprising two symmetrical layers of origami DNA,
wherein the shape of each DNAFile is stabilized by
a) adding a sequence of six or more thymidine resides (poly(T)) to one end of the the data oligonucleotides;
b) decreasing the length of staple oligonucleotides located near sheet corners to less than 100 nucleotides, or less than 50 nucleotides, to allow for flexibility during the folding process;
c) introducing intentional gaps or missing base pairs within the scaffold DNA strand/staple folded structure (i.e. “skips”) near the center-line of the folded multi-layered sheet; or
d) any combination of a) through c).

4. The library of claim 3 wherein said data oligonucleotides have a length of about 30 to 200 nucleotides, and the first and second primer binding sequences, and the sequence complementary to a nucleic acid sequence of said single stranded DNA scaffold, are each independently 10 to 20 nucleotides in length.

5. The library of claim 1 wherein
i) said first primer binding sequence is located at the 5′ terminus of said data oligonucleotides and said second primer binding sequence is located at the 3′ terminus of said data oligonucleotides; or
ii) said nucleic acid sequence of the data oligonucleotide that is complementary to said single stranded DNA scaffold is 5′ to said first primer binding sequence, or 3′ to said second primer binding sequence.

6. The library of claim 1 wherein each member of said plurality of origami folded DNAFiles comprises a different single stranded DNA scaffold.

7. The library of claim 1 wherein
i) each member of said plurality of origami folded DNAFiles has a unique shape; or
ii) each origami folded DNAFile further comprises a linked unique nucleic acid barcode construct; or
iii) both i) and ii).

8. The library of claim 1 wherein each origami folded DNAFile further comprises a unique nucleic acid barcode construct linked to the origami DNAFile via base-pairing, wherein said base-pairing that links the nucleic acid barcode construct with the origami DNAFile occurs between
i) a single-stranded non-complementary nucleic acid sequence of one or more of said staple oligonucleotides and a complementary sequence linked to the nucleic acid barcode construct; or
ii) a single-stranded non-complementary nucleic acid sequence extending from the 5′ or 3′ end of the single-stranded DNA scaffold and a complementary sequence linked to the nucleic acid barcode construct.

9. The library of claim 8 , wherein the nucleic acid barcode construct is linked to the DNAFile by a high affinity, non-covalent bond interaction between a biotin molecule linked to the 5′ and/or the 3′ end of the nucleic acid barcode construct and a molecule that binds to biotin, said molecule being linked to the DNAFile.

10. A library comprising a plurality of origami folded DNA data storage files (DNAFiles), each of said DNAFiles comprising
a single stranded DNA scaffold; and
a plurality of single stranded DNA staple oligonucleotides that each bind through complementary base pairing with two non-contiguous nucleic acid sequences of the DNA scaffold, wherein said staple oligonucleotides cause the DNA scaffold to fold into a two or three dimensional shape having a first surface;
a plurality of data oligonucleotides, said data oligonucleotides comprising a sequence complementary to a nucleic acid sequence of said single stranded DNA scaffold, a nucleic acid sequence that encodes digital information, and a first primer binding sequence and a second primer binding sequence, wherein the first primer binding sequence is 5′ to the digital information encoding nucleic acid sequence, the second primer binding sequence is 3′ to the digital information encoding nucleic acid sequence, wherein said plurality of data oligonucleotides are localized to said first surface, wherein the individual DNAFiles differ from one another based on the nucleic acid sequences of the plurality of data oligonucleotides bound to the DNA scaffold of each DNAFile wherein the data oligonucleotides of each individual origami folded DNAFile of said library further comprise an identical set of PCR binding sequences for preselected PCR primers, where the PCR binding sequences differ between the data oligonucleotides of each respective DNAFile of the library.

11. A method of retrieving digital data stored in DNA, said method comprising
providing the library of origami folded DNAFiles according to claim 1 ;
denaturing a folded origami DNAFile of said library to at least partially disrupt the hybridized duplex between the single stranded staple oligonucleotides, data oligonucleotides and the DNA scaffold;
conducting PCR amplification on select nucleic acid sequences of said denatured DNA scaffold and data oligonucleotides to produce amplicons;
reannealing the staple oligonucleotides and data oligonucleotides with the DNA scaffold to reconstitute the folded origami DNAFile;
separating the amplicons from the reconstituted folded origami DNAFile;
returning the reconstituted folded origami DNAFile to the library; and
sequencing the amplicons to retrieve digital data encoded by the DNAFile.

12. The method of claim 11 wherein said denaturing step completely releases all staple oligonucleotides and data oligonucleotides as free single stranded nucleic acids.

13. The method of claim 11 wherein the amplicons are separated from the reconstituted folded origami DNAFiles
i) via gel electrophoresis; or
ii) via size exclusion chromatography.

14. The method of claim 11 further comprising the step of confirming the correct size and shape of the reconstituted folded origami DNA scaffold prior to returning the reconstituted folded origami DNA scaffold to the library.

15. The method of claim 14 further comprising the step of selecting one or more individual origami folded DNAFiles from the other origami folded DNAFiles of said library and conducting the denaturing step only on the selected origami folded DNAFiles.

16. The method of claim 15

Assignees

• Battelle Memorial Institute

Inventors

• Gupta Cherry
• Bartling Craig M
• Spurbeck Rachel R
• Duong Anthony D
• Ha James
• Pedrozo Miguel D
• Andrioff Nickolas R

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• G16B15/10

  Nucleic acid folding · CPC title

• G06N3/123

  DNA computing · CPC title

• G16B30/20

  Sequence assembly · CPC title

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title