Ligation of 2 ', 3 '-cyclic phosphate RNAs for the identification of microRNA binding sites
OPEN ACCESS
Loading...
Author / Producer
Date
2021-01
Publication Type
Journal Article
ETH Bibliography
yes
Citations
Altmetric
OPEN ACCESS
Data
Rights / License
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.
Permanent link
Publication status
published
External links
Editor
Book title
Journal / series
Volume
595 (2)
Pages / Article No.
230 - 240
Publisher
Wiley
Event
Edition / version
Methods
Software
Geographic location
Date collected
Date created
Subject
2 ',3 '-cyclic phosphate; CLIP; ligation; microRNA; RtcB
Organisational unit
03760 - Hall, Jonathan / Hall, Jonathan
Notes
Funding
169612 - Chemical Approaches to Functionalize Human microRNAs (SNF)