Chemical Labeling and Affinity Capture of Inosine-Containing RNAs Using Acrylamidofluorescein

Bioconjugate Chemistry
Steve D KnutsonJennifer M Heemstra

Abstract

Adenosine-to-inosine (A-to-I) RNA editing is a widespread and conserved post-transcriptional modification, producing significant changes in cellular function and behavior. Accurately identifying, detecting, and quantifying these sites in the transcriptome is necessary to improve our understanding of editing dynamics, its broader biological roles, and connections with diseases. Chemical labeling of edited bases coupled with affinity enrichment has enabled improved characterization of several forms of RNA editing. However, there are no approaches currently available for pull-down of inosines. To address this need, we explore acrylamide as a labeling motif and report here an acrylamidofluorescein reagent that reacts with inosine and enables enrichment of inosine-containing RNA transcripts. This method provides improved sensitivity in the detection and identification of inosines toward a more comprehensive transcriptome-wide analysis of A-to-I editing. Acrylamide derivatization is also highly generalizable, providing potential for the labeling of inosine with a wide variety of probes and affinity handles.

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Citations

May 30, 2020·Current Protocols in Chemical Biology·Steve D Knutson, Jennifer M Heemstra
May 20, 2020·Chemistry : a European Journal·Steve D KnutsonJennifer M Heemstra
Nov 7, 2019·Wiley Interdisciplinary Reviews. RNA·Rachel Netzband, Cara T Pager
Jan 15, 2021·Current Opinion in Structural Biology·Steve D Knutson, Jennifer M Heemstra
Feb 24, 2021·Critical Reviews in Biochemistry and Molecular Biology·Yasemin YoluçStefanie Kellner
Feb 29, 2020·Journal of the American Chemical Society·Steve D KnutsonJennifer M Heemstra
Jun 15, 2021·Advanced Biology·Maik SchauerteKatharina Höfer
Jul 15, 2021·Chemical Society Reviews·David BarteeJordan L Meier
Sep 14, 2019·Organic Letters·Ying LiRobert C Spitale

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