Ligation of 2′, 3′-cyclic phosphate RNAs for the identification of microRNA binding sites

FEBS Letters

article

Christian Berk¹  Yuluan Wang¹  Artur Laski¹  Stylianos Tsagkris¹  Jonathan Hall¹ 

[1]Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences

关键词: 2′;    3′-cyclic phosphate;    CLIP;    ligation;    microRNA;    RtcB;   

DOI  :  10.1002/1873-3468.13976

来源: John Wiley & Sons Ltd.

【 摘 要 】

Identifying the targetome of a microRNA is key for understanding its functions. Cross-linking and immunoprecipitation (CLIP) methods capture native miRNA-mRNA interactions in cells. Some of these methods yield small amounts of chimeric miRNA-mRNA sequences via ligation of 5′-phosphorylated RNAs produced during the protocol. Here, we introduce chemically synthesized microRNAs (miRNAs) bearing 2′-, 3′-cyclic phosphate groups, as part of a new CLIP method that does not require 5′-phosphorylation for ligation. We show in a system that models miRNAs bound to their targets, that addition of recombinant bacterial ligase RtcB increases ligation efficiency, and that the transformation proceeds via a 3′-phosphate intermediate. By optimizing the chemistry underlying ligation, we provide the basis for an improved method to identify miRNA targetomes.

【 授权许可】

Unknown