Compositions of matter for detection assays

1. A composition of matter comprising a ribonucleoprotein (RNP) complex and a blocked nucleic acid molecule, wherein the blocked nucleic acid molecule is represented by Formula I, wherein Formula I in the 5′-to-3′ direction comprises:

A-(B-L) J -C-M-T-D;

wherein A is 0-15 nucleotides in length;

B is 4-12 nucleotides in length;

L is 3-25 nucleotides in length;

J is an integer between 1 and 10;

C is 4-15 nucleotides in length;

M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L) J -C and T-D are separate nucleic acid strands;

T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C;

D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and

wherein the blocked nucleic acid molecule comprises a sequence complementary to a gRNA; and wherein the ribonucleoprotein complex comprises a nucleic acid-guided nuclease and the gRNA; and wherein the nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity;

wherein the nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease;

wherein the blocked nucleic acid molecule comprises a modified nucleoside or nucleotide, and 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.

2. The composition of matter of claim 1 , wherein the nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease.

3. The composition of matter of claim 1 , wherein the nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity.

4. The composition of matter of claim 1 , wherein the nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

5. The composition of matter of claim 1 , wherein:

(a) T of Formula I comprises at least 80% sequence complementarity to B and C; and/or

(b) D of Formula I comprises at least 80% sequence complementarity to A.

6. The composition of matter of claim 1 , wherein the blocked nucleic acid molecule does not comprise a PAM sequence.

7. The composition of matter of claim 1 , wherein the blocked nucleic acid molecule comprises a PAM sequence disposed between first and second sequences, wherein the first sequence is 5′ to the PAM sequence.

8. The composition of matter of claim 1 , wherein the blocked nucleic acid molecule is a blocked primer molecule.

9. The composition of matter of claim 1 , further comprising a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by the ribonucleoprotein complex.

10. The composition of matter of claim 9 , wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.

11. The composition of matter of claim 10 , wherein the detectable signal is the fluorescent signal.

12. The composition of matter of claim 10 , wherein the detectable signal is the chemiluminescent signal.

13. The composition of matter of claim 10 , wherein the detectable signal is the colorimetric signal.

14. The composition of matter of claim 10 , wherein the detectable signal is the other optical signal.

15. The composition of matter of claim 9 , wherein the reporter moiety comprises a modified nucleoside or nucleotide.

16. The composition of matter of claim 15 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.

17. The composition of matter of claim 1 , further comprising a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by the ribonucleoprotein complex.

18. The composition of matter of claim 17 , wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.

19. The composition of matter of claim 18 , wherein the detectable signal is the fluorescent signal.

20. The composition of matter of claim 18 , wherein the detectable signal is the chemiluminescent signal.

21. The composition of matter of claim 18 , wherein the detectable signal is the radioactive signal.

22. The composition of matter of claim 18 , wherein the detectable signal is the colorimetric signal.

23. The composition of matter of claim 18 , wherein the detectable signal is the other optical signal.

24. The reaction mixture of claim 17 , wherein the reporter moiety comprises a modified nucleoside or nucleotide.

25. The composition of matter of claim 24 , 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.