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.

Ligation of 2', 3'-cyclic phosphate RNAs for the identification of microRNA binding sites / Berk, Christian; Wang, Yuluan; Laski, Artur; Tsagkris, Stylianos; Hall, Jonathan. - In: FEBS LETTERS. - ISSN 0014-5793. - 595:2(2021), pp. 230-240. [10.1002/1873-3468.13976]

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

Tsagkris, Stylianos;Hall, Jonathan
2021

Abstract

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.
2021
2′,3′-cyclic phosphate; CLIP; RtcB; ligation; microRNA
01 Pubblicazione su rivista::01a Articolo in rivista
Ligation of 2', 3'-cyclic phosphate RNAs for the identification of microRNA binding sites / Berk, Christian; Wang, Yuluan; Laski, Artur; Tsagkris, Stylianos; Hall, Jonathan. - In: FEBS LETTERS. - ISSN 0014-5793. - 595:2(2021), pp. 230-240. [10.1002/1873-3468.13976]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1724370
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