Tuning cascade assay kinetics via molecular design

A reaction mixture for detecting target nucleic acids of interest comprising:

• first ribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise a first nucleic acid-guided nuclease and first gRNAs;
• wherein the first gRNAs comprise a sequence complementary to the target nucleic acids 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 nucleic acid targets 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 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 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 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 the standard free energy changes for the 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 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 primer molecule;
• a polymerase and a plurality of nucleotides;
• and
• a plurality of reporter moieties, wherein the plurality of reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP1 an RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample.