Detection assays
Presented is a nucleic acid-guided nuclease cascade assay that can detect one or more target nucleic acids of interest of interest without the need for amplifying the target nucleic acids of interest. The nucleic acid-guided nuclease cascade assays utilize signal amplification mechanisms comprising various components including nucleic acid-guided nucleases and guide RNAs (gRNAs) forming ribonucleoprotein complexes, blocked primer molecules, template molecules, polymerases and reporter moieties.
1. A cascade assay method for detecting a target nucleic acid of interest in a sample comprising the steps of:
(a) providing a reaction mixture comprising:
i. a first ribonucleoprotein (RNP) complex (RNP1) comprising a first Cas12a nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity;
ii. a second ribonucleoprotein complex (RNP2) comprising a second Cas12a nucleic acid-guided nuclease and a second gRNA; wherein the second gRNA is complementary to synthesized activating molecules and the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity;
iii. a plurality of template molecules comprising a sequence having homology to the second gRNA and a primer binding domain;
iv. a plurality of blocked primer molecules comprising a sequence complementary to the primer binding domain of the plurality of template molecules and wherein the blocked primer molecules cannot be extended by a polymerase;
v. a Phi29 or T7 DNA polymerase and dNTPs;
wherein when blocked, the blocked primer molecules cannot bind to the template molecules and be extended by the polymerase and wherein when unblocked, the blocked primer molecules can bind to the primer binding domain of the template molecules and be extended by the polymerase to produce synthesized activating molecules;
(b) contacting the reaction mixture with the sample under conditions that allow target nucleic acids of interest in the sample to bind to the first gRNA;
wherein:
upon binding of the target nucleic acid of interest, the RNP1 becomes active cleaving at least one of the blocked primer molecules, thereby producing at least one unblocked primer molecule that can be extended by the polymerase;
at least one unblocked primer molecule binds to one of the template molecules and is extended by the polymerase and nucleotides to form at least one synthesized activating molecule having a sequence complementary to the second gRNA;
at least one synthesized activating molecule binds to the second gRNA, and RNP2 becomes active cleaving at least one further blocked primer molecule;
and
(c) detecting the cleavage products of step (a), thereby detecting the target nucleic acid of interest in the sample.
2. The method of claim 1 , wherein the polymerase exhibits 3′→5′ exonuclease activity.
3. The method of claim 1 , wherein the blocked primer molecule comprises a modified nucleoside or nucleotide.
4. The method of claim 3 , wherein the modified nucleoside or nucleotide comprises a locked nucleic acid (LNA), peptide nucleic acid (PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond.
5. The method of claim 1 , wherein the template molecules do not comprise a PAM sequence complement.
6. The method of claim 1 , wherein the template molecules comprise a PAM sequence complement.
7. The method of claim 1 , further comprising a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and produces a detectable signal upon cleavage by the ribonucleoprotein complex.
8. The method of claim 7 , wherein the reporter moiety comprises a modified nucleoside or nucleotide.
9. The method of claim 8 , wherein the modified nucleoside or nucleotide comprises a locked nucleic acid (LNA), peptide nucleic acid (PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond.
10. The method of claim 7 , wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.
11. The method of claim 10 , wherein the detectable signal is the fluorescent signal.
12. The method of claim 10 , wherein the detectable signal is the chemiluminescent signal.
13. The method of claim 10 , wherein the detectable signal is the colorimetric signal.
14. The method of claim 1 , comprising about 1 fM to about 1 mM of the RNP2.
15. The method of claim 1 , wherein a K d of the blocked primer molecules to the template molecules is about 10 6 -fold greater or more than the K d of an unblocked primer molecule resulting from unblocking of the blocked primer molecules.
16. The method of claim 15 , wherein a K d of the blocked primer molecules to the template molecules is about 10 7 -fold greater or more than the K d of an unblocked primer molecule resulting from unblocking of the blocked primer molecules.
17. The method of claim 16 , wherein a K d of the blocked primer molecules to the template molecules is about 10 8 -fold greater or more than the K d of an unblocked primer molecule resulting from unblocking of the blocked primer molecules.
18. The method of claim 17 , wherein a K d of the blocked primer molecules to the template molecules is about 10 10 -fold greater or more than the K d of an unblocked primer molecule resulting from unblocking of the blocked primer molecules.
19. The method of claim 1 , wherein there are at least two different first ribonucleoprotein complexes, wherein said different first ribonucleoprotein complexes comprise a first gRNA complementary to a different target nucleic acid of interest.
20. The method of claim 19 , wherein there are at least ten different first ribonucleoprotein complexes, wherein said different first ribonucleoprotein complexes comprise a first gRNA complementary to a different target nucleic acid of interest.
21. The method of claim 20 , wherein there are at least 100 different first ribonucleoprotein complexes wherein different first ribonucleoprotein complexes comprise first gRNAs complementary to different target nucleic acids of interest.