Chimeric Oligonucleotides for Ligation-Enhanced Nucleic Acid Detection, Methods and Compositions Therefor

Abstract

Ligation-enhanced nucleic acid detection assay embodiments for detection of RNA or DNA are described. The assay embodiments rely on ligation of chimeric oligonucleotide probes to generate a template for amplification and detection. The assay embodiments are substantially independent of the fidelity of a polymerase for copying compromised nucleic acid. Very little background amplification is observed and as few as 1000 copies of target nucleic acid can be detected. Method embodiments are particularly adept for detection of RNA from compromised samples such as formalin-fixed and paraffin-embedded samples. Heavily degraded and cross-linked nucleic acids of compromised samples, in which classic quantitative real time PCR assays typically fail to adequately amplify signal, can be reliably detected and quantified.

1 . A method for detecting a target nucleic acid in a sample, comprising:
 a. contacting the sample with at least one set of chimeric oligonucleotide probes for a time and under conditions suitable to form an annealed product, wherein the at least one set of chimeric oligonucleotide probes comprises at least one first chimeric oligonucleotide probe and at least one second chimeric oligonucleotide probe;
 wherein the at least one first chimeric oligonucleotide probe comprises, in a 5′ to 3′ direction:
 a primer-specific portion comprising an amplification primer nucleotide sequence; and 
 a target-specific portion, the target-specific portion having:
 complementarity to a 3′ portion of a preselected sequence of the target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, 
 at least one nucleotide analog at one of the six 5′-most nucleotides wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide, and 
 3′-OH and 2′-OR groups on the 3′-terminal nucleotide, wherein R comprises H or C 1 -C 3  alkyl; 
 
 
 wherein the at least one second chimeric oligonucleotide probe comprises, in a 5′ to 3′ direction:
 a target-specific portion having:
 a 5′-terminal nucleotide comprising a 5′-phosphate group, 
 complementarity to a 5′ portion of the preselected sequence of the target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, and 
 a primer-specific portion comprising an amplification primer nucleotide sequence; 
 
 
 wherein, when the at least one first and the at least one second chimeric oligonucleotide probes are annealed to the target nucleic acid, the 3′ hydroxyl group of the first chimeric oligonucleotide probe is positioned immediately adjacent to the 5′ phosphate group of the second chimeric oligonucleotide probe; 
   b. contacting the annealed product with a polypeptide having double-strand dependent ligase activity for a time and under conditions suitable to form a ligated product; and   c. detecting the target nucleic acid in the sample by detecting the ligated product, or a surrogate thereof.   
     
     
         2 . The method of  claim 1 , further comprising adding a single-strand specific ribonuclease prior to b, in or during b, or prior to c. 
     
     
         3 . The method of  claim 2 , wherein the target nucleic acid is from a compromised sample. 
     
     
         4 . The method of  claim 2 , wherein the target nucleic acid comprises DNA. 
     
     
         5 . The method of  claim 2 , wherein the target nucleic acid comprises RNA. 
     
     
         6 . The method of  claim 5 , wherein the RNA is siRNA or miRNA. 
     
     
         7 . The method of  claim 3 , wherein the compromised sample comprises an FFPE sample. 
     
     
         8 . The method of  claim 2 , wherein the polypeptide having double-strand dependent ligase activity comprises an RNA ligase and the RNA ligase comprises T4 Rnl2 ligase, DraRnl, KVP40 Rnl2, TbREL1, TbREL2, LtREL1, LtREL2, or an enzymatically active mutant or variant thereof. 
     
     
         9 . The method of  claim 8 , wherein the RNA ligase comprises T4 Rnl2 ligase. 
     
     
         10 . The method of  claim 2 , wherein the ligated product comprises an addressable sequence selected from a detector probe sequence or a hybridization capture sequence. 
     
     
         11 . The method of  claim 10 , wherein the detector probe sequence comprises a 5′-nuclease probe sequence and the detector probe sequence includes a sequence that corresponds to a 3′-portion of the target-specific sequence of the first chimeric oligonucleotide probe taken together with a 5′ portion of the target-specific sequence of the second chimeric oligonucleotide probe. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . A set of chimeric oligonucleotide probes, the probe set comprising:
 a first chimeric oligonucleotide probe, comprising, in a 5′ to 3′ direction:
 a primer-specific portion comprising an amplification primer nucleotide sequence; and 
 a target-specific portion, the target-specific portion having:
 complementarity to a 3′ portion of a preselected sequence of a target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, 
 at least one nucleotide analog at one of the six 5′-most nucleotides wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide, and 
 3′-OH and 2′-OR groups on the 3′-terminal nucleotide, wherein R comprises H or C 1 -C 3  alkyl; and 
 
   a second chimeric oligonucleotide probe comprising, in a 5′ to 3′ direction:
 a target-specific portion having:
 a 5′-terminal nucleotide comprising a 5′-phosphate group, 
 complementarity to a 5′ portion of the preselected sequence of the target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, and 
 
 a primer-specific portion comprising an amplification primer nucleotide sequence; 
 wherein, when the first and second chimeric oligonucleotide probes are annealed to the target nucleic acid, the 3′ hydroxyl group of the first chimeric oligonucleotide probe is positioned immediately adjacent to the 5′ phosphate group of the second chimeric oligonucleotide probe. 
   
     
     
         15 . The set of chimeric oligonucleotide probes of  claim 14 , wherein the target-specific portion of the second chimeric oligonucleotide probe comprises at least one nucleotide analog at one of the six 3′-most nucleotides, wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide. 
     
     
         16 . The set of chimeric oligonucleotide probes of  claim 15 , wherein the 3′-terminal and the 3′-penultimate nucleotides of the first chimeric oligonucleotide probe comprise non-modified ribonucleotides. 
     
     
         17 . The set of chimeric oligonucleotide probes of  claim 15 , wherein two, three, four, five, or six of the six 5′-most nucleotides of the target-specific portion of the first chimeric oligonucleotide probe comprise a nucleotide analog, and wherein two, three, four, five, or six of the six 3′-most nucleotides of the target-specific portion of the second chimeric oligonucleotide probe comprise a nucleotide analog, wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide. 
     
     
         18 . The set of chimeric oligonucleotide probes of  claim 17 , wherein the nucleotide analogs of the first chimeric oligonucleotide probe are contiguous. 
     
     
         19 . The set of chimeric oligonucleotide probes of  claim 17 , wherein the nucleotide sequence of the target-specific portion of the first chimeric oligonucleotide probe together with the nucleotide sequence of the target-specific portion of the second chimeric oligonucleotide probe is designed to anneal across an exon junction of the target nucleic acid. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . A set of at least three oligonucleotide probes, comprising:
 a 5′-most chimeric oligonucleotide probe comprising, in a 5′ to 3′ direction:
 a primer-specific portion comprising an amplification primer nucleotide sequence; and 
 a target-specific portion, the target-specific portion having:
 complementarity to a 3′-portion of a preselected sequence of a target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, 
 at least one nucleotide analog at one of the six 5′-most nucleotides wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide, and 
 3′-OH and 2′-OR groups on the 3′-terminal nucleotide, wherein R comprises H or C 1 -C 3  alkyl; 
 
   a 3′-most oligonucleotide probe comprising, in a 5′ to 3′ direction:
 a target-specific portion having:
 a 5′-terminal nucleotide comprising a 5′-phosphate group, 
 complementarity to a 5′ portion of the preselected sequence of the target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, and 
 
 a primer-specific portion comprising an amplification primer nucleotide sequence; and 
   a medial probe consisting essentially of:
 a target-specific portion having:
 a 5′-terminal nucleotide comprising a 5′-phosphate group, 
 complementarity to a portion of the preselected sequence of the target nucleic acid intermediate the 5′-most and 3′-most oligonucleotide probes when annealed to target nucleic acid, 
 a length of 6 nucleotides to 44 nucleotides, 
 primarily non-modified ribonucleotides wherein any nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide, and 
 3′-OH and 2′-OR groups on the 3′-terminal nucleotide, wherein R comprises H or C 1 -C 3  alkyl; 
 
   wherein, when the 5′-most, medial, and 3′-most probes are annealed to the target nucleic acid, the 3′ hydroxyl group of the 5′-most chimeric oligonucleotide probe is positioned immediately adjacent to the 5′ phosphate group of the medial probe, and the 3′ hydroxyl group of the medial probe is positioned immediately adjacent to the 5′ phosphate group of the 3′-most chimeric oligonucleotide probe.   
     
     
         24 - 25 . (canceled)