COMPOSITIONS AND METHODS FOR DETECTION OF A NUCLEIC ACID
Described herein are methods, devices, systems, and compositions for detecting a target nucleic acid using a programmable nuclease and a signal amplifier. Such methods, devices, systems, and compositions can comprise using signal amplifiers, such as catalytic oligonucleotides, which can be activated by programmable nucleases and be configured to cleave a reporter molecule upon activation. Signals can be generated and detected upon cleavage of reporter molecules.
1 . A composition comprising a signal amplifier, a programmable nuclease, and a guide nucleic acid that hybridizes to a segment of a target nucleic acid.
2 . The composition of claim 1 , wherein the signal amplifier comprises an enzyme.
3 . The composition of claim 1 , wherein the signal amplifier comprises a catalytic oligonucleotide.
4 . The composition of claim 3 , wherein the catalytic oligonucleotide has a circular structure.
5 . The composition of claim 3 , wherein the catalytic oligonucleotide comprises a programmable nuclease cleavage site.
6 . (canceled)
7 . The composition of claim 3 , further comprising a blocker oligonucleotide.
8 . (canceled)
9 . (canceled)
10 . The composition of claim 7 , wherein the blocker oligonucleotide comprises a programmable nuclease cleavage site, a catalytic oligonucleotide recognition site, or a combination thereof.
11 . (canceled)
12 . The composition of claim 3 , wherein the catalytic oligonucleotide comprises an enzyme.
13 . The composition of claim 3 , wherein the catalytic oligonucleotide comprises a DNAzyme or a ribozyme.
14 .- 17 . (canceled)
18 . The composition of claim 1 , wherein the programmable nuclease is a type VI CRISPR/Cas effector protein or a type V CRISPR/Cas effector protein.
19 .- 27 . (canceled)
28 . The composition of claim 1 , further comprising the target nucleic acid.
29 . The composition of claim 28 , wherein the target nucleic acid is a target RNA or a target DNA.
30 . (canceled)
31 . The composition of claim 1 , further comprising a reporter molecule.
32 . The composition of claim 31 , wherein the reporter molecule is configured to generate a signal upon cleavage by a catalytic oligonucleotide, the programmable nuclease, or both.
33 . (canceled)
34 . (canceled)
35 . The composition of claim 1 , wherein the programmable nuclease is a first programmable nuclease and the composition further comprises a second programmable nuclease.
36 . A composition comprising a first signal amplifier, a second signal amplifier, a programmable nuclease, and a guide nucleic acid that hybridizes to a segment of a target nucleic acid.
37 . The composition of claim 36 , wherein the first signal amplifier comprises a first enzyme and the second signal amplifier comprises a second enzyme.
38 . The composition of claim 36 , wherein the first signal amplifier comprises a first catalytic oligonucleotide and the second signal amplifier comprises a second catalytic oligonucleotide.
39 .- 72 . (canceled)
73 . A method of nucleic acid detection comprising:
(a) contacting a sample to a composition comprising a plurality of reporter molecules and the composition of claim 1 ; and
(b) assaying for a signal produced by or indicative of cleavage one or more of the reporter molecules.
74 .- 80 . (canceled)
81 . A method of nucleic acid detection comprising:
(a) contacting a sample comprising a plurality of nucleic acids to a composition comprising a plurality of reporter molecules, a programmable nuclease complex comprising a programmable nuclease coupled to a guide nucleic acid that hybridizes to a segment of a target nucleic acid, and a signal amplifier;
(b) when the target nucleic acid is present in the plurality of nucleic acids, activating the programmable nuclease complex by hybridizing the target nucleic acid, or an amplicon thereof, to the guide nucleic acid;
(c) activating the signal amplifier with the activated programmable nuclease complex, wherein the activated signal amplifier is configured to cleave at least a reporter molecule of the plurality of reporter molecules; and
(d) assaying for a signal produced by or indicative of cleavage of the reporter molecule.