Signal boost cascade assay

Abstract

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide signal boost upon detection of target nucleic acids of interest in less than one minute and in some instances instantaneously at ambient temperatures down to 16° C. or less, without amplification of the target nucleic acids yet allowing for massive multiplexing, high accuracy and minimal non-specific signal generation.

We claim: 
     
         1 . A method for identifying a target nucleic acid of interest in a sample in one minute or less at 16° C. comprising the steps of:
 providing a reaction mixture comprising:
 first ribonucleoprotein (RNP1) complexes (RNP1s) each comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; 
 second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; 
 a plurality of blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP2 complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein optionally the molar ratio of the blocked nucleic acid molecules is at least equal to the molar ratio of the second ribonucleoprotein complexes, and wherein optionally the blocked nucleic acid molecules each comprise at least one bulky modification; 
 and wherein one of the following conditions is met: 1) providing blocked nucleic acid molecules and ribonucleoprotein complexes where the molar ratio of the blocked nucleic acid molecules is equal to or exceeds the molar ratio of the ribonucleoprotein complexes, 2) the blocked nucleic acid molecules each comprise at least one bulky modification, or 3) the RNP2 comprises at least one variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved; and 
 
 contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1; wherein upon binding of the target nucleic acid of interest RNP1 becomes active initiating trans-cleavage of at least one of the plurality of blocked nucleic acid molecules thereby producing at least one unblocked nucleic acid molecule, wherein the at least one unblocked nucleic acid molecule binds to RNP2 initiating trans-cleavage of at least one further blocked nucleic acid molecule; and 
 detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample in one minute or less. 
 
     
     
         2 . The method of  claim 1 , wherein the reaction mixture further comprises reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample. 
     
     
         3 . The method of  claim 2 , wherein the reporter moieties are not coupled to the blocked nucleic acid molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected. 
     
     
         4 . The method of  claim 2 , wherein the reporter moieties are coupled to the blocked nucleic acid molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected. 
     
     
         5 . The method of  claim 1 , wherein the reaction mixture comprises blocked nucleic acid molecules with bulky modifications and wherein the bulky modifications are about 1 nm in size. 
     
     
         6 . The method of  claim 6 , wherein the reaction mixture comprises blocked nucleic acid molecules with bulky modifications and wherein the bulky modifications are about 0.7 nm in size. 
     
     
         7 . The method of  claim 1 , wherein blocked nucleic acid molecules include bulky modifications and wherein there are two bulky modifications with one bulky modification located on the 5′ end of the blocked nucleic acid molecule and one bulky modification located on the 3′ end of the blocked nucleic acid molecule, and where the 5′ and 3′ ends comprising the two bulky modifications are less than 11 nm from one another. 
     
     
         8 . The method of  claim 1 , wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is on a 5′ end of blocked nucleic acid molecules. 
     
     
         9 . The method of  claim 1 , wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is on a 3′ end of the blocked nucleic acid molecules. 
     
     
         10 . The method of  claim 1 , wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is between two internal nucleic acid residues of the blocked nucleic acid molecules. 
     
     
         11 . The method of  claim 1 , wherein the RNP2s comprise a variant nuclease and the variant nuclease comprises at least one mutation to the PAM-acting domain selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs. 
     
     
         12 . The method of  claim 11 , wherein there are at least two mutations to the PAM-acting domain selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs. 
     
     
         13 . The method of  claim 12 , wherein there are at least three mutations to the PAM-acting domain selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs. 
     
     
         14 . The method of  claim 1 , wherein the RNP2s comprise a variant nuclease and the variant nuclease comprises at least one mutation to the PAM-acting domain of the variant nucleic acid-guided nuclease and wherein the at least one mutation is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2; mutations to amino acid residues K534, Y538 and R591 in relation to SEQ ID NO:3; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:4; mutations to amino acid residues K579, N583 and K635 in relation to SEQ ID NO:5; mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:6; from mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:7; mutations to amino acid residues K617, N621 and K678 in relation to SEQ ID NO:8; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:9; mutations to amino acid residues K569, N573 and K625 in relation to SEQ ID NO:10; mutations to amino acid residues K562, N566 and K619 in relation to SEQ ID NO:11; mutations to amino acid residues K645, N649 and K732 in relation to SEQ ID NO:12; mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:13; mutations to amino acid residues K592, N596 and K653 in relation to SEQ ID NO:14; or mutations to amino acid residues K521, N525 and K577 in relation to SEQ ID NO:15. 
     
     
         15 . The method of  claim 1 , wherein the RNP2s comprise a variant nucleic acid-guided nuclease comprising at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules and wherein single stranded DNA is cleaved at least two times faster than double stranded DNA is cleaved. 
     
     
         16 . The method of  claim 1 , wherein the plurality of blocked nucleic acid molecules and the RNP2s are at a molar concentration of at least 2 blocked nucleic acids to 1 RNP2 in the reaction mixture. 
     
     
         17 . The method of  claim 1 , wherein the target nucleic acid molecule of interest is of bacterial or viral origin. 
     
     
         18 . The method of  claim 1 , wherein the target nucleic acid molecule of interest is from a human or other animal. 
     
     
         19 . The method of  claim 18 , wherein the sample is selected from blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, a transudate, an exudate, or fluid obtained from a joint, or a swab of skin or mucosal membrane surface. 
     
     
         20 . The method of  claim 21 , wherein the sample is a blood sample from a transplant patient and the target nucleic acid molecule is a donor-derived genomic sequence. 
     
     
         21 . The method of  claim 21 , wherein the sample is a blood sample from a transplant patient and the target nucleic acid molecules are a hemoglobin S gene and a hemoglobin C gene. 
     
     
         22 . The method of  claim 20 , wherein the target nucleic acid molecule is a pathogen that infects livestock. 
     
     
         23 . The method of  claim 1 , wherein the sample is an environmental sample. 
     
     
         24 . The method of  claim 23 , wherein the sample is selected from the group of a soil sample, an air sample, and a water sample. 
     
     
         25 . The method of  claim 24 , wherein the sample is a sewer sample. 
     
     
         26 . The method of  claim 1 , wherein the target nucleic acid molecule is a pathogen used as a bioweapon. 
     
     
         27 . The method of  claim 20 , wherein the target nucleic acid is a human biomarker. 
     
     
         28 . The method of  claim 27 , wherein the human biomarker is a cancer biomarker. 
     
     
         29 . The method of  claim 1 , wherein there are at least ten target nucleic acid molecules of interest in the sample. 
     
     
         30 . The method of  claim 29 , wherein there are at least twenty target nucleic acid molecules of interest in the sample.