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 a blocking nucleic acid molecule represented by Formula IV, wherein Formula IV in the 5′-to-3′ direction comprises: T-D-M-A-Lp-C; wherein T is 17-31 nucleotides in length; D is 0-15 nucleotides in length; M is 1-25 nucleotides in length; A is 0-15 nucleotides in length and comprises a sequence complementary to D; and L is 3-25 nucleotides in length; p is 0 or 1; C is 4-15 nucleotides in length and comprises a sequence complementary to T; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.
1. A composition of matter comprising:
a ribonucleoprotein complex (RNP) comprising a nucleic acid-guided nuclease and a gRNA complementary to synthesized activating molecules;
a plurality of template molecules comprising a sequence having homology to the gRNA and a primer binding domain;
a plurality of blocked primer molecules comprising a sequence complementary to the primer binding domain of the plurality of template molecules and having a Formula IV, wherein Formula IV in the 5′-to-3′ direction comprises:
T-D-M-A-L p -C;
wherein T is 17-31 nucleotides in length;
D is 0-15 nucleotides in length;
M is 1-25 nucleotides in length;
A is 0-15 nucleotides in length and comprises a sequence complementary to D; and
L is 3-25 nucleotides in length;
p is 0 or 1;
C is 4-15 nucleotides in length and comprises a sequence complementary to T; and
a 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 template molecules and be extended by the polymerase to produce synthesized activating 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 exhibits both cis- and trans-cleavage activity.
5. 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.
6. The composition of matter of claim 1 , wherein:
(a) A of Formula IV comprises at least 80% sequence complementarity to D; and/or
(b) C of Formula IV comprises at least 80% sequence complementarity to T.
7. The composition of matter of claim 1 , wherein the blocked primer molecule comprises a modified nucleoside or nucleotide.
8. The composition of matter of claim 7 , 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.
9. The composition of matter of claim 1 , wherein the template molecules do not comprise a PAM sequence complement.
10. The composition of matter of claim 1 , wherein the template molecules comprise a PAM sequence complement.
11. 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 produces a detectable signal upon cleavage by the ribonucleoprotein complex.
12. The composition of matter of claim 11 , wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.
13. The composition of matter of claim 12 , wherein the detectable signal is the fluorescent signal.
14. The composition of matter of claim 12 , wherein the detectable signal is the chemiluminescent signal.
15. The composition of matter of claim 12 , wherein the detectable signal is the colorimetric signal.
16. The composition of matter of claim 11 , wherein the reporter moiety comprises a modified nucleoside or nucleotide.
17. The composition of matter of claim 16 , 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.
18. The composition of matter of claim 1 , comprising about 1 fM to about 1 mM of the RNP.
19. The composition of matter 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.
20. The composition of matter of claim 19 , 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.
21. The composition of matter of claim 20 , 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.
22. The composition of matter of claim 21 , 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.
23. The composition of matter of claim 1 , wherein the polymerase exhibits 3′→5′ exonuclease activity.
24. The composition of matter of claim 1 , wherein the polymerase is Phi29 polymerase.
25. The composition of matter of claim 1 , wherein the polymerase is a Type I, Type II, or Type III DNA polymerase.