Compositions of matter for detection assays
The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula III in the 5′-to-3′ direction comprises: T-D-M-A-(B-L) J -C; wherein T is 17-135 nucleotides in length; D is 0-10 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L) J -C are separate nucleic acid strands; A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D; B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.
1. A composition of matter comprising a ribonucleoprotein complex and a blocked nucleic acid molecule, wherein the blocked nucleic acid molecule is represented by Formula III,
wherein
Formula III in the 5′-to-3′ direction comprises:
T-D-M-A-(B-L) J -C;
wherein T is 17-135 nucleotides in length;
D is 0-10 nucleotides in length;
M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L) J -C are separate nucleic acid strands;
A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;
B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
L is 3-25 nucleotides in length;
J is an integer between 1 and 10;
C is 4-15 nucleotides in length;
wherein the blocked nucleic acid molecules comprise a sequence complementary to a gRNA; wherein the ribonucleoprotein complex comprises a nucleic acid-guided nuclease and the gRNA; wherein the nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; and wherein a K d of the blocked nucleic acid molecules to the RNP is about 10 5 -fold greater or more than the K d of an unblocked nucleic acid molecules resulting from unblocking of the blocked nucleic acid molecules.
2. The composition of matter of claim 1 , wherein the nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease.
3. 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.
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) A of Formula III comprises at least 80% sequence complementarity to D; and/or
6 (b) B of Formula III comprises at least 80% sequence complementarity to T.
6. The composition of matter of claim 1 , wherein the blocked nucleic acid molecule comprises a modified nucleoside or nucleotide.
7. The composition of matter of claim 6 , 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.
8. The composition of matter of claim 1 , wherein the blocked nucleic acid molecule does not comprise a PAM sequence.
9. 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.
10. 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.
11. The composition of matter of claim 10 , wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.
12. The composition of matter of claim 11 , wherein the detectable signal is the fluorescent signal.
13. The composition of matter of claim 11 , wherein the detectable signal is the chemiluminescent signal.
14. The composition of matter of claim 11 , wherein the detectable signal is the colorimetric signal.
15. The composition of matter of claim 10 , 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 17 , wherein the reporter moiety comprises a modified nucleoside or nucleotide.
24. The composition of matter of claim 23 , 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.
25. The composition of matter of claim 1 , wherein the composition of matter comprises about 1 fM to about 1 mM of the RNP.
26. The composition of matter of claim 1 , wherein a K d of the blocked nucleic acid molecules to the RNP is about 10 6 -fold greater or more than the K d of an unblocked nucleic acid molecule resulting from unblocking of the blocked nucleic acid molecules.
27. The composition of matter of claim 26 , wherein a K d of the blocked nucleic acid molecules to the RNP is about 10 7 -fold greater or more than the K d of an unblocked nucleic acid molecule resulting from unblocking of the blocked nucleic acid molecules.
28. The composition of matter of claim 27 , wherein a K d of the blocked nucleic acid molecules to the RNP is about 10 8 -fold greater or more than the K d of an unblocked nucleic acid 29 molecule resulting from unblocking of the blocked nucleic acid molecules.
29. The composition of matter of claim 28 , wherein a K d of the blocked nucleic acid molecules to the RNP is about 10 10 -fold greater or more than the K d of an unblocked nucleic acid molecule resulting from unblocking of the blocked nucleic acid molecules.