OTTR-CLASH: improved biochemical and bioinformatic identification of Argonaute 2-mediated microRNA-target RNA interactions

Authors

Kaufman, P. D., Liu, H., Hu, K., Ferguson, L., Collins, K., Zhu, L. J., Pederson, T.

Abstract

Various methods have detected miRNA-target interactions via immunoprecipitation of UV-crosslinked Argonaute ribonucleoprotein complexes, followed by intermolecular ligation of bound miRNAs to target strands, forming chimeric RNAs. To date, these methods have relied on conventional viral reverse transcriptases (RTs) to generate cDNAs for sequencing. However, crosslinked RNAs often retain adducts after purification, which can make them poor templates for viral RTs. Here, we adapted OTTR (Ordered Two-Template Relay) techniques to generate cDNAs from Ago2-bound RNAs. OTTR makes use of a modified retroelement-encoded RT, which is strongly processive even on templates with modifications or adducts. We show that this OTTR-CLASH method increases the frequency of generating chimeric RNAs compared to previous methods. We also developed an improved bioinformatic pipeline for analysis of these data, and we use this to catalog miRNA-target interactions not previously described in the literature.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 16 Jul 2026.

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