Methods and compositions for molecular authentication

Patent metadata

Field	Value
Publication number	US-12142104-B2
Application number	US-201917040041-A
Country	US
Kind code	B2
Filing date	Mar 21, 2019
Priority date	Mar 22, 2018
Publication date	Nov 12, 2024
Grant date	Nov 12, 2024

Abstract

Provided herein, in some embodiments, are molecular authentication methods, systems, and compositions.

First claim

What is claimed is:

1. A method of detecting a target nucleic acid, comprising
contacting the target nucleic acid with an authenticating identifier composition, wherein the target nucleic acid comprises a first strand comprising domain x* and domain a, and the authenticating identifier composition comprises:
(a) a second strand comprising domain a*, domain x, domain b*, and a first molecule of a quencher-fluorophore pair, wherein domain a* binds to domain a, and domain x binds to domain x*;
(b) a third strand comprising a second molecule of the quencher-fluorophore pair, domain b, and domain x*, wherein domain b binds to domain b*; and
(c) a fourth strand comprising domain b and domain x*;
wherein binding of the second strand to the first strand produces a detectable signal, wherein the detectable signal is an optical signal.

2. The method of claim 1 , wherein the first strand of the target nucleic acid comprises domain x* and domain a in a 5′ to 3′ direction; the second strand comprises domain a*, domain x, domain b*, and the first molecule of a quencher-fluorophore pair in a 5′ to 3′ direction; the third strand comprises the second molecule of the quencher-fluorophore pair, domain b, and domain x* in a 5′ to 3′ direction; and the fourth strand comprises domain b and domain x* in a 5′ to 3′ direction.

3. The method of claim 1 , wherein the first strand of the target nucleic acid comprises domain x* and domain a in a 3′ to 5′ direction; the second strand comprises domain a*, domain x, domain b*, and the first molecule of a quencher-fluorophore pair in a 3′ to 5′ direction; the third strand comprises the second molecule of the quencher-fluorophore pair, domain b, and domain x* in a 3′ to 5′ direction; and the fourth strand comprises domain b and domain x* in a 3′ to 5′ direction.

4. The method of claim 1 , wherein a concentration of the fourth strand in the authenticating identifier composition is greater than a combined concentration of the second and third strands.

5. The method of claim 4 , wherein the concentration of the fourth strand in the authenticating identifier composition is at least 2-fold greater than the combined concentration of the second and third strands.

6. The method of claim 1 , wherein the first, second, third, and/or fourth strand is 10-30 nucleotides in length.

7. The method of claim 1 , wherein at least one of the strands of the authenticating identifier composition is immobilized to a surface.

8. The method of claim 7 , wherein the surface is a capillary strip or paper-based material.

9. The method of claim 8 , wherein the nanoparticle is a gold nanoparticle.

10. The method of claim 7 , wherein the surface is a surface of a nanoparticle.

11. The method of claim 10 , wherein the first catalytic hairpin strand comprises domain b, the loop domain, domain b* and domain a* in a 5′ to 3′ direction; and the second catalytic hairpin strand comprises domain c, the loop domain, domain c* and domain b* in a 5′ to 3′ direction.

12. The method of claim 10 , wherein the first catalytic hairpin strand comprises domain b, the loop domain, domain b* and domain a* in a 3′ to 5′ direction; and the second catalytic hairpin strand comprises domain c, the loop domain, domain c* and domain b* in a 3′ to 5′ direction.

13. The method of claim 1 , wherein at least one of the strands of the authenticating identifier composition is immobilized to a test substrate comprising (i) a source region, (ii) a test region comprising the immobilized strand(s), and (iii) a control region.

14. The method of claim 1 , wherein at least one of the strands of the authenticating identifier composition is immobilized to a test substrate comprising (i) a source region comprising enzyme-linked strands, (ii) a test region comprising the immobilized strand(s) and an embedded enzyme substrate, and (iii) a control region comprising an embedded enzyme substrate and immobilized control strands that bind to the enzyme-linked strands.

15. The method of claim 1 , wherein contacting of the target nucleic acid with the authenticating identifier composition is performed at a temperature of about 20 to 25° C.

16. A method of detecting a target nucleic acid, comprising
(a) contacting the target nucleic acid with a first catalytic hairpin strand that comprises domain b, a loop domain, domain b* and domain a* and a second catalytic hairpin strand that comprises domain c, a loop domain, domain c* and domain b*, wherein domain b binds to domain b*, and domain c binds to domain c*; and
(b) removing the first and second catalytic hairpin strands from the target nucleic acid;
(c) combining the removed catalytic hairpin strands with (i) a primer comprising domain a, wherein domain a binds to domain a*, (ii) a strand displacing polymerase, and (iii) dNTPs, and producing a key strand comprising domain a, domain b, and domain c; and
(d) authenticating the key strand using a molecular lock composition, wherein the molecular lock composition comprises a lock strand comprising domain a*, domain b*, and domain c*, and wherein binding of the lock strand to the key strand produces a detectable signal, wherein the detectable signal is an optical signal.

17. The method of claim 16 , wherein the first key strand of comprises domain a and domain x in a 5′ to 3′ direction; and the second key strand comprises domain b and domain y in a 5′ to 3′ direction.

18. The method of claim 16 , wherein the first key strand comprises domain a and domain x in a 3′ to 5′ direction; and the second key strand comprises domain b and domain y in a 3′ to 5′ direction.

19. A method of detecting a target nucleic acid, comprising
(a) contacting the target nucleic acid with a first key strand that comprises domain a and domain x and a second key strand that comprises domain b and domain y; and
(b) removing the first and second key strands from the target nucleic acid;
(c) combining the removed key strands with a molecular lock composition, wherein the molecular lock composition comprises (i) a first lock strand comprising, in a 5′ to 3′ direction, a first molecule of a quencher-fluorophore pair, domain y*, domain b*, domain x*, and domain a*, (ii) a second lock strand comprising, in a 5′ to 3′ direction, domain x and domain b, and (iii) a third lock strand comprising, in a 5′ to 3′ direction, domain y and a second molecule of the quencher-fluorophore pair, wherein domain a binds to domain a*, domain x binds to domain x*, domain b binds to domain b*, and domain y binds to domain y*, wherein binding of the first and second key strands to the first lock strand produces a detectable signal, wherein the detectable signal is an optical signal.

Assignees

• Harvard College

Inventors

• Yin Peng
• Kishi Jocelyn Yoshiko
• Schaus Thomas E
• Kim Youngeun

Classifications

• C12Q2563/185

  Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals · CPC title

• C12Q1/6813

  Hybridisation assays · CPC title

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• G07D7/14 Primary

  using chemical means · CPC title