US 12,247,205 B2
Tuning cascade assay kinetics via molecular design
Ashish Pandey, San Diego, CA (US); Ariana Mostafa, San Diego, CA (US); Jacob Berger, 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 Aug. 15, 2024, as Appl. No. 18/805,534.
Application 18/805,534 is a continuation of application No. 18/586,417, filed on Feb. 23, 2024, granted, now 12,104,158.
Application 18/586,417 is a continuation of application No. 18/518,914, filed on Nov. 24, 2023, granted, now 11,946,052, issued on Apr. 2, 2024.
Application 18/518,914 is a continuation of application No. 18/208,262, filed on Jun. 10, 2023, granted, now 11,884,922, issued on Jan. 30, 2024.
Application 18/208,262 is a continuation of application No. 18/204,329, filed on May 31, 2023, granted, now 11,859,182, issued on Jan. 2, 2024.
Application 18/204,329 is a continuation of application No. 18/076,262, filed on Dec. 6, 2022, granted, now 11,820,983, issued on Nov. 21, 2023.
Claims priority of provisional application 63/402,055, filed on Aug. 29, 2022.
Claims priority of provisional application 63/289,112, filed on Dec. 13, 2021.
Prior Publication US 2024/0401051 A1, Dec. 5, 2024
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)] 28 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;
a plurality of template molecules comprising a sequence complementary to the second gRNA and comprising a primer binding domain;
a plurality of tunable blocked primer molecules comprising a sequence complementary to the primer binding domain on the template molecules, wherein each tunable blocked primer molecule comprises: a first region recognized by the RNP2 complexes; 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 free energy of the plurality of tunable blocked primer molecules at 25° C. is at most about −5 kcal/mol when the following formula is used to calculate free energy for each base pair: ΔG° (T)=(ΔH°−TΔS°) cal mol−1, and total ΔG° is given by: ΔG° (total)=Σini ΔG° (i)+ΔG°(init with term G·C)+ΔG°(init with term A·T)+ΔG° (sym), where ΔG° (i) are standard free energy changes for the 10 possible Watson-Crick NNs, n; is the number of occurrences of each nearest neighbor, i, and ΔG° (sym) equals+0.43 kcal/mol if a 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 unblocked primer molecules; and
a polymerase and a plurality of nucleotides;
contacting the reaction mix with the sample under conditions that allow non-nucleic acid targets of interest in the sample to bind to the RNP1s, wherein:
(i) upon binding of the target nucleic acid of interest, the RNP1s become active, cleaving at least one of the blocked primer molecules, thereby producing at least one unblocked primer molecule that can be extended by the polymerase;
(ii) at least one unblocked primer molecule binds to one of the template molecules and is extended by the polymerase and nucleotides to form at least one synthesized activating molecule having a sequence complementary to the second gRNA;
(iii) at least one synthesized activating molecule binds to the second gRNA, and the RNP2s become active, cleaving at least one further blocked primer molecule;
allowing a cascade reaction to continue; and detecting the activated RNP2s, thereby detecting the target nucleic acid of interest in the sample.