FEBS Letters | |
Ligation of 2′, 3′-cyclic phosphate RNAs for the identification of microRNA binding sites | |
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
【 预 览 】
Files | Size | Format | View |
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RO202302050001955ZK.pdf | 1824KB | download |