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 the presence of one or more target RNAs in a sample, said method comprising:
a) ligating an adapter or linker oligonucleotide to the 3′ end of said one or more target RNAs to produce one or more extended target polynucleotides; b) circularizing said one or more extended target polynucleotides by ligating the 5′-end of the one or more extended target polynucleotides to its 3′- end; c) synthesizing by rolling circle amplification complementary multimeric nucleic acids (MNAs) comprising multiple repeats of sequences that are complementary to said one or more circularized extended polynucleotides; and d) detecting said MNAs, thereby detecting the presence of said one or more target RNAs in said sample.