CPC C12Q 1/6823 (2013.01) [C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12Q 1/682 (2013.01); C12Q 1/6806 (2013.01); C12N 2310/20 (2017.05); C12N 2310/315 (2013.01); C12N 2310/321 (2013.01); C12N 2310/3231 (2013.01); C12N 2320/10 (2013.01)] | 28 Claims |
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 a Kd of the blocked nucleic acid molecules to the RNP is about 105-fold greater or more than the Kd of an unblocked nucleic acid molecule resulting from unblocking of the blocked nucleic acid molecules.
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