US 11,946,052 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 Nov. 24, 2023, as Appl. No. 18/518,914.
Application 18/518,914 is a continuation of application No. 18/208,262, filed on Jun. 10, 2023, granted, now 11,884,922.
Application 18/208,262 is a continuation of application No. 18/204,329, filed on May 31, 2023, granted, now 11,859,182.
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.
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 composition of matter for detecting a ribonucleic acid target of interest in a sample comprising:
first ribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise an RNA-specific nucleic acid-guided nuclease and a first gRNA; wherein the first gRNA comprises a sequence complementary to the ribonucleic acid target of interest, and wherein the RNA-specific nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity;
second ribonucleoprotein complexes (RNP2s), wherein the RNP2s comprise Cas12a nucleic acid-guided nuclease and a second gRNA that is not complementary to the nucleic acid target of interest, and wherein the Cas12a nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity;
a plurality of tunable blocked nucleic acid molecules, wherein each tunable blocked nucleic acid 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 the free energy of each tunable blocked nucleic acid molecule primer 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)=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 10 possible Watson-Crick NNs, ni 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 an unblocked nucleic acid molecule; and
a plurality of reporter moieties, wherein the plurality of reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP1s and RNP2s to identify the presence of one or more nucleic acid targets of interest in the sample.