Compositions and methods of a nuclease chain reaction for nucleic acid detection

Patent metadata

Field	Value
Publication number	US-2024141412-A1
Application number	US-202117926441-A
Country	US
Kind code	A1
Filing date	May 18, 2021
Priority date	May 19, 2020
Publication date	May 2, 2024
Grant date	—

Abstract

Described herein are nucleic acid detection compositions and systems comprising an internal nuclease chain reaction (NCR) for signal amplification and methods of using these NCR-containing compositions and systems.

First claim

1 . A nucleic acid detection system comprising:
i) a reporter molecule comprising a detectable label, wherein the detectable label is released for detection upon cleavage of the reporter molecule; ii) a primary activator complex comprising a first recognizing complex, and; iii) an inactive secondary complex wherein;
a) the first recognizing complex recognizes one or more primary activators in a sample, wherein;
b) upon recognition of the primary activator, the primary activator complex is activated and is able to act on the reporter molecule to release the detectable label, and;
c) the activated primary activator complex is able to act on a component of an inactive second recognizing complex to activate the second recognizing complex to become an activated signal amplifier, wherein;
d) said activated signal amplifier is able to act on the reporter molecule to release the detectable label, and;
e) said activated signal amplifier is able to act on a component of an inactive second recognizing complex to activate the second recognizing complex to become an activated signal amplifier such that a feed-forward loop is initiated.

2 . A nucleic acid detection system comprising:
a reporter molecule comprising a detectable label, wherein the detectable label is released for detection upon cleavage of the reporter molecule; a primary activator complex comprising a first Cas-effector enzyme programmed with a first guide RNA, wherein the first guide RNA recognizes one or more primary activators in a sample, wherein upon hybridization of the first guide RNA to the primary activator, the primary activator complex is activated and is a non-specific nuclease that cleaves the reporter molecule and releases the detectable label; and a signal amplifier comprising a second Cas-effector enzyme and a second guide RNA, wherein activation of the primary activation complex results in the activation of one or more activator sequences that are recognized by the second guide RNA, wherein upon hybridization of the second guide RNA to the activator sequence the signal amplifier complex is activated and is a non-specific nuclease that cleaves the reporter molecule and releases the detectable label.

3 . The nucleic acid detection system of claim 1 , wherein the first and/or second Cas-effector enzymes comprise a non-specific RNase and/or a DNase when activated.

4 . The nucleic acid detection system of claim 1 , wherein the first and/or second Cas-effector enzymes comprises one or more Cas13 proteins, one or more Cas12 proteins, one or more Cas14 proteins, one or more Csm6 proteins, and/or one or more Csx1 proteins, optionally one or more proteins as shown in any one of SEQ ID NOs: 115 through 268.

5 . The nucleic acid detection system of claim 1 , wherein the first and/or second Cas-effectors comprise one or more Cas13 proteins, optionally one or more Cas13 proteins as shown in any one of SEQ ID NOs: 115 to 135.

6 . The nucleic acid detection system of claim 1 , wherein the first and/or second Cas-effectors comprise one or more Cas13d proteins.

7 . The nucleic acid detection system of claim 1 , wherein the first and/or second Cas-effectors comprise one or more Cas12 proteins, one or more Cas13 proteins, one or more Cas14 proteins, and/or one or more Csm6 proteins in any combination.

8 . The nucleic acid detection system of claim 1 , wherein the first and second Cas-effector enzymes comprise one or more of the same or different proteins.

9 - 10 . (canceled)

11 . The nucleic acid detection system of claim 1 , wherein the reporter molecule comprises a quencher operably linked to the detectable label, optionally wherein the detectable label comprises one or more fluorescent molecule.

12 . The nucleic acid detection system of claim 1 , wherein the reporter molecule comprises an oligonucleotide linking the quencher and the fluorophore, and the oligonucleotide comprises a caged structure, optionally having or causing a stem-loop structure.

13 . The nucleic acid detection system of claim 1 , wherein the reporter molecule is complexed with a trans cage molecule, optionally having or causing a stem loop structure.

14 . The nucleic acid detection system of claim 1 , wherein the reporter molecule the detectable label comprises one or more fluorescent dyes.

15 . The nucleic acid detection system of claim 1 , wherein the activator complex is caged, optionally having or causing a stem-loop structure.

16 . The nucleic acid detection system of claim 1 , wherein the activator complex further comprises an oligonucleotide sequence wherein the oligonucleotide sequence comprises modified nucleotide bases.

17 . The nucleic acid detection system of claim 1 , wherein the activator complex further comprises an oligonucleotide sequence wherein the oligonucleotide sequence comprises both RNA and DNA bases.

18 . The nucleic acid detection system of claim 1 , wherein the guide RNA is caged, optionally comprising or causing a stem-loop structure.

19 . The nucleic acid detection system of claim 1 , wherein one or more of the amplifier sequences and/or one or both of the guide RNAs are caged, optionally wherein the cage comprises or causes one or more structures such as a loop structure and/or a modification to one or more of the amplifier sequences comprising one or more locked nucleic acid (LNA) or moieties and/or 2′-OMe RNA.

20 . The nucleic acid detection system of claim 1 , wherein one or more of the amplifier sequences comprising caging structures on their 3′ and/or 5′ ends.

21 . The nucleic acid detection system of claim 1 , further comprising trans caging molecules.

22 . The nucleic acid detection system of claim 1 , wherein the one or both of the first and second guide RNAs and/or one or more of the amplifier sequences are modified to allow conditional interaction with the Cas-effector enzyme during the optimal time frame.

23 . The nucleic acid detection system of claim 1 , wherein the one or more amplifier sequences comprise poly U and/or poly A sequences, optionally A 4 -U n , A 5 -U n and A 6 -U n sequences.

24 . The nucleic acid detection system of claim 1 , wherein the target sequence and/or amplifier sequence is(are) 100% complementary to first and/or second guide RNAs.

25 . The nucleic acid detection system of claim 1 , wherein the target sequence and/or amplifier sequence is(are) not 100% complementary to first and/or second guide RNAs.

26 . The nucleic acid detection system of claim 1 , wherein the target sequence is DNA or RNA from one or more mammals, viruses, bacteria, or fungi.

27 . The nucleic acid detection system of claim 1 , wherein the target sequence is in an RNA virus.

28 . The nucleic acid detection system of claim 1 , wherein the target sequence is in a coronavirus, optionally a SARS-Cov-2 coronavirus.

29 . The nucleic acid detection system of claim 1 , wherein the sample is a biological or environmental sample.

30 . The nucleic acid detection system of claim 1 , wherein the biological sample comprises blood, saliva, urine, biopsy, plasma, serum, bronchoalveolar lavage, sputum, a fecal sample, cerebrospinal fluid, a fine needle aspirate, a buccal swab, a cervical swab, a nasal swab, interstitial fluid, synovial fluid, nasal discharge, tears, buffy coat, a mucous membrane sample, or an epithelial cell sample collected from the

Assignees

• Univ California

Inventors

• Savage David
• Hsu Patrick
• Prywes Noam
• Charles Emeric J
• Knott Gavin J
• Desmarais John James
• Kim Shineui
• Dugan Eli
• Lukarska Maria
• Chandrasekaran Sita S
• Lammers Nicholas C
• Liu Tina Y
• Mok Amanda
• Lareau Liana
• Thornton Brittney Wai-Ling

Classifications

• C12Q1/6818 Primary

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6816 Primary

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title