Selective enrichment of A-to-I edited transcripts from cellular RNA using Endonuclease V

Immunoprecipitation enrichment has significantly improved the sensitivity and accuracy of detecting RNA modifications in the transcriptome. However, there are no existing methods for selectively isolating adenosine-to-inosine (A-to-I) edited RNAs. Here we show that Escherichia coli Endonuclease V (eEndoV), an inosine-cleaving enzyme, can be repurposed to bind and isolate A-to-I edited transcripts from cellular RNA through adjustment of cationic conditions. While Mg 2+ is required for eEndoV catalysis, it has also been shown that similar levels of Ca 2+ instead promote binding of inosine without cleavage. Leveraging these properties, we observe that Ca 2+ -supplemented eEndoV is highly specific for inosine in RNA and exhibits low nanomolar binding affinity. We then demonstrate EndoVIPER ( Endo nuclease V i nosine precipitation e n r ichment) as a facile and robust method to isolate A-to-I edited transcripts from cellular RNA. We envision the use of this approach as a straightforward and cost-effective strategy to enrich edited RNAs and detect A-to-I sites with improved sensitivity and fidelity.