RNase H-Based Assays Utilizing Modified RNA Monomers

Abstract

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3′ end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

1 : A method of detection of a target nucleic acid sequence within a sample using an oligonucleotide ligation reaction, wherein the method comprises the steps of:
 (a) contacting the sample with an acceptor oligonucleotide and a donor oligonucleotide, wherein the acceptor oligonucleotide and/or donor oligonucleotide comprises an RNase H cleavable domain,
 (i) wherein if the acceptor oligonucleotide comprises the RNase H cleavable domain, then there is a blocking group at or near the 3′-end of the acceptor oligonucleotide, wherein the blocking group inhibits the ligation reaction, or 
 (ii) wherein if the donor oligonucleotide comprises the RNase H cleavable domain, then the donor oligonucleotide either has a 5′ hydroxyl group, or a 5′ phosphate and a blocking group at or near the 5′-end of the donor oligonucleotide, wherein the blocking group inhibits the ligation reaction; 
   (b) hybridizing the acceptor and donor oligonucleotides to the target DNA sequence to form a double-stranded substrate;   (c) cleaving the hybridized acceptor and/or donor oligonucleotide with an RNase H enzyme at the RNase H cleavable site within or adjacent to the cleavage domain; and   (d) ligating the acceptor oligonucleotide to the donor oligonucleotide with a ligation agent.   
     
     
         2 : The method of  claim 1 , wherein the RNase H cleavage domain comprises a single RNA residue. 
     
     
         3 : The method of  claim 1 , wherein the RNase H cleavage domain lacks an RNA residue. 
     
     
         4 : The method of  claim 3 , wherein the cleavage domain comprises one or more 2′-modified nucleosides. 
     
     
         5 : The method of  claim 1 , wherein the RNase H enzyme is an RNase H2 enzyme. 
     
     
         6 : The method of  claim 1 , wherein the ligation agent is a DNA ligase enzyme. 
     
     
         7 : The method of  claim 6 , wherein the DNA ligase enzyme is a thermostable DNA ligase enzyme. 
     
     
         8 : The method of  claim 7 , wherein the thermostable DNA ligase enzyme is a hot-start DNA ligase enzyme having reduced activity at lower temperatures. 
     
     
         9 : The method of  claim 8 , wherein the DNA ligase enzyme inherently has lower activity at reduced temperatures or is reversibly inactivated either by chemical modification or by a blocking antibody. 
     
     
         10 : The method of  claim 1 , wherein the blocking group is attached 5′ of the 3′ terminal residue of the acceptor oligonucleotide. 
     
     
         11 : The method of  claim 1 , wherein the blocking group includes one or more abasic residues or modified nucleosides. 
     
     
         12 : The method of  claim 11 , wherein the abasic residue is a C3 spacer. 
     
     
         13 : A method of detection of a target nucleic acid sequence within a sample using an oligonucleotide ligation reaction, wherein the method comprises the steps of:
 (a) contacting the sample with a self-ligating oligonucleotide, wherein the acceptor end and/or donor end of said oligonucleotide comprises an RNase H cleavable domain,
 (i) wherein if the acceptor end comprises the RNase H cleavable domain, then there is a blocking group at or near its 3′-end, wherein the blocking group inhibits the ligation reaction, or 
 (ii) wherein if the donor end comprises the RNase H cleavable domain, then the donor end either has a 5′ hydroxyl group, or a 5′ phosphate and a blocking group at or near its 5′-end, wherein the blocking group inhibits the ligation reaction; 
   (b) hybridizing the self-ligating oligonucleotide to the target DNA sequence to form a double-stranded substrate;   (c) cleaving the hybridized acceptor and/or donor end with an RNase H enzyme at the RNase H cleavable site within or adjacent to the cleavage domain; and   (d) ligating the acceptor and donor ends of the oligonucleotide with a ligation agent to form a circular DNA molecule.   
     
     
         14 : The method of  claim 13 , wherein the RNase H cleavage domain comprises a single RNA residue. 
     
     
         15 : The method of  claim 13 , wherein the RNase H cleavage domain lacks an RNA residue. 
     
     
         16 : The method of  claim 15 , wherein the cleavage domain comprises one or more 2′-modified nucleosides. 
     
     
         17 : The method of  claim 13 , wherein the RNase H enzyme is an RNase H2 enzyme 
     
     
         18 : The method of  claim 13 , wherein the ligation agent is a DNA ligase enzyme. 
     
     
         19 : The method of  claim 18 , wherein the DNA ligase enzyme is a thermostable DNA ligase enzyme. 
     
     
         20 : The method of  claim 19 , wherein the thermostable DNA ligase enzyme is a hot-start DNA ligase enzyme having reduced activity at lower temperatures. 
     
     
         21 : The method of  claim 20 , wherein the DNA ligase enzyme inherently has lower activity at reduced temperatures or is reversibly inactivated either by chemical modification or by a blocking antibody. 
     
     
         22 : The method of  claim 13 , wherein the blocking group is attached 5′ of the 3′ terminal residue of the self-ligating oligonucleotide. 
     
     
         23 : The method of  claim 13 , wherein the blocking group includes one or more abasic residues or modified nucleosides. 
     
     
         24 : The method of  claim 23 , wherein the abasic residue is a C3 spacer.