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 kit for the ligation of the donor and accepter ends of a single oligonucleotide in the presence of a target nucleic acid sequence comprising:
 (a) an oligonucleotide in which either its donor end, its accepter end, or both its donor and accepter ends comprise a cleavage domain and a blocking group preventing ligation of its 5′ phosphate group to its 3′ OH group;   (b) a cleaving agent;   (c) a ligation agent; and   (d) optionally, an instruction manual for ligating the accepter and donor ends of the oligonucleotide in the presence of a target nucleic acid sequence.   
     
     
         2 . The kit of  claim 1 , wherein the cleavage domain is an RNase H cleavable domain. 
     
     
         3 . The kit of  claim 2 , wherein the cleavage domain comprises a single RNA residue. 
     
     
         4 . The kit of  claim 2 , wherein the cleavage domain lacks an RNA residue. 
     
     
         5 . The kit of  claim 4 , wherein the cleavage domain comprises one or more 2′-modified nucleosides. 
     
     
         6 . The kit of  claim 1 , wherein the cleaving agent is an RNase H enzyme. 
     
     
         7 . The kit of  claim 6 , wherein the RNase H enzyme is an RNase H2 enzyme. 
     
     
         8 . The kit of  claim 1 , wherein the ligation agent is a DNA ligase enzyme. 
     
     
         9 . The kit of  claim 8 , wherein the DNA ligase enzyme is a thermostable DNA ligase. 
     
     
         10 . The kit of  claim 9 , wherein the thermostable DNA ligase is a hot-start DNA ligase having reduced activity at lower temperatures. 
     
     
         11 . The kit of  claim 10 , wherein the DNA ligase inherently has lower activity at reduced temperatures or is reversibly inactivated either by chemical modification or by a blocking antibody. 
     
     
         12 . The kit of  claim 1 , wherein the blocking group is attached 5′ of the 3′ terminal residue of the accepter group on the oligonucleotide. 
     
     
         13 . The kit of  claim 1 , wherein the blocking group includes one or more abasic residues or modified nucleosides. 
     
     
         14 . The kit of  claim 13 , wherein the abasic residue is a C3 spacer.