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