Methods and compositions for detection of small RNAs

摘要

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5′-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2′OH and 2′-OMe at their 3′ ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2′OH and 2′-OMe at their 3′ ends while allowing us to distinguish these two forms from each other.

主权项

1. A method of detecting a target RNA in a sample comprising:
a) circularizing said target RNA in said sample by ligating the 5′-end of said target RNA to its 3′-end to produce a circularized target RNA, wherein said circularized target RNA is from 10-100 nucleotides in length; b) synthesizing a multimeric nucleic acid (MNA) comprising multiple repeats of sequences that are complementary to said target RNA by rolling circle amplification; wherein said synthesizing comprises:
i. hybridizing said circularized target RNA with an oligonucleotide reverse transcriptase (RT) primer specific for said target RNA; andii. enzymatically extending said oligonucleotide RT primer by a polymerase comprising reverse transcriptase activity to produce said MNA; and c) amplifying the MNA with a forward primer and a reverse primer to produce an amplicon, wherein:
i. the 3′-proximal region of the forward primer is complementary to a first portion of the multiple repeats within said MNA;ii. the 3′-proximal region of the reverse primer corresponds in sequence to a second portion of the multiple repeats within said MNA; andiii. the forward primer and reverse primer can form a duplex with 3′ overhangs, wherein said duplex is shorter than said target sequence by at least 2 nucleotides; and d) detecting the amplicon, thereby detecting the target RNA in the sample.