	US 11,884,922 B1
	Tuning cascade assay kinetics via molecular design
Ariana Mostafa, San Diego, CA (US); Jacob Berger, San Diego, CA (US); Ashish Pandey, San Diego, CA (US); and Anurup Ganguli, San Diego, CA (US)
Assigned to VedaBio, Inc., San Diego, CA (US)
Filed by VedaBio, Inc., San Diego, CA (US)
Filed on Jun. 10, 2023, as Appl. No. 18/208,262.
Application 18/208,262 is a continuation of application No. 18/204,329, filed on May 31, 2023.
Application 18/204,329 is a continuation of application No. 18/076,262, filed on Dec. 6, 2022, granted, now 11,820,983.
Claims priority of provisional application 63/289,112, filed on Dec. 13, 2021.
Claims priority of provisional application 63/402,055, filed on Aug. 29, 2022.
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/113 (2010.01); C12N 15/11 (2006.01)

CPC C12N 15/113 (2013.01) [C12N 15/111 (2013.01); C12N 2310/20 (2017.05); C12N 2310/315 (2013.01); C12N 2310/322 (2013.01)]

30 Claims

1. A method for detecting a nucleic acid target of interest in a sample comprising the steps of:

providing reaction mix comprising:

first ribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise a first nucleic acid-guided nuclease and a first gRNA; wherein the first gRNA comprises a sequence complementary to the nucleic acid target of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity;

second ribonucleoprotein complexes (RNP2s), wherein the RNP2s comprise a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest, and wherein the second nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity; and

a plurality of tunable blocked nucleic acid molecules, wherein each tunable blocked nucleic acid molecule comprises: a first region recognized by the RNP2 complex; one or more second regions not complementary to the first region forming at least one loop; and one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the free energy of each tunable blocked primer molecule at 25° C. is at most about −5 kcal/mol when the following formula is used to calculate the free energy for each base pair: ΔG°(T)=(ΔH°−TΔS°)cal mol⁻¹, and total ΔG°is given by: ΔG°(total)=Σ_in_iΔG°(i)+ΔG°(init with term G·C)+ΔG°(init with term A·T)+ΔG°(sym), where ΔG°(i) are the standard free energy changes for the 10 possible Watson-Crick NNs), n_iis the number of occurrences of each nearest neighbor, i, and ΔG°(sym) equals+0.43 kcal/mol if the duplex is self-complementary and zero if it is non-self-complementary, and wherein cleavage of the one or more second regions results in dehybridization of the one or more the third regions from the first region, resulting in an unblocked nucleic acid molecule;

contacting the reaction mix with the sample under conditions that allow non-nucleic acid targets of interest in the sample to bind to the RNP1, wherein:

upon binding of the target nucleic acid of interest to the RNP1, the RNP1 becomes active trans-cleaving at least one tunable blocked nucleic acid molecule, thereby producing at least one unblocked nucleic acid molecule that can complex with the RNP2; and

upon binding of the at least one unblocked nucleic acid molecule to the RNP2, the RNP2 becomes active trans-cleaving at least one more tunable blocked nucleic acid molecule;

allowing a cascade reaction to continue; and

detecting the unblocked nucleic acid molecule, thereby detecting the target nucleic acid of interest in the sample.