# AvaII senses m6A and inosine sites and enables targeted nanopore direct RNA-sequencing

## TL;DR

A new method uses the enzyme AvaII to improve nanopore RNA sequencing by targeting specific RNA modifications and generating new sequencing starting points.

## Contribution

AvaII is shown to cleave RNA at GGACC sites and sense m6A and inosine modifications, enabling targeted sequencing.

## Key findings

- AvaII cleaves RNA at GGACC sites when hybridized to a complementary DNA oligo.
- AvaII does not cleave RNA with m6A or inosine in the central position.
- AvaII cleavage products are compatible with targeted nanopore direct RNA-sequencing.

## Abstract

Nanopore direct RNA-sequencing is the first commercialized method to sequence native RNA directly, thus preserving RNA modifications. With the current technology, sequencing is initiated from the 3′end. While for relatively short polyadenylated RNAs, full coverage is obtained, the 5′end of many long RNAs is not sufficiently covered resulting in a substantial 3′bias. We aimed to cleave such RNAs in a sequence-specific manner in order to generate new unique 3′ends that can be targeted by custom adapters. We identified the DNA endonuclease AvaII as a candidate enzyme. AvaII was originally described to cleave double-stranded DNA at GGWCC sites, where W is an A or T. Here, we show that AvaII cleaves also long RNAs in GGACC contexts, if hybridized to a complementary DNA oligo. Furthermore, we provide evidence that AvaII cleavage of RNA is modification sensitive and does not cleave RNA with m6A or inosine in the central position. We propose AvaII as “methylation sensor” for the bona fide DRACH recognition motif GGACC of the m6A writer complex. Finally, we show that AvaII cleavage products are accessible to targeted Nanopore direct RNA-sequencing.