Chemical Probe-Based Nanopore Sequencing to Selectively Assess the RNA Modifications

Soundhar Ramasamy, Vinodh J. Sahayasheela, Surbhi Sharma, Zutao Yu, Takuya Hidaka, Li Cai, Vaijayanthi Thangavel, Hiroshi Sugiyama, Ganesh N. Pandian

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Nanopore direct RNA sequencing (dRNA-Seq) reads reveal RNA modifications through consistent error profiles specific to a modified nucleobase. However, a null data set is required to identify actual RNA modification-associated errors for distinguishing it from confounding highly intrinsic sequencing errors. Here, we reveal that inosine creates a signature mismatch error in dRNA-Seq reads and obviates the need for a null data set by harnessing the selective reactivity of acrylonitrile for validating the presence of actual inosine modifications. Selective reactivity of acrylonitrile toward inosine altered multiple dRNA-Seq parameters like signal intensity and trace value. We also deduced the stoichiometry of inosine modification through deviation in signal intensity and trace value using this chemical biology approach. Furthermore, we devised Nano ICE-Seq, a protocol to overcome the low coverage issue associated with direct RNA sequencing. Taken together, our chemical probe-based approach may facilitate the knockout-free detection of disease-associated RNA modifications in clinical scenarios.

Original languageEnglish (US)
Pages (from-to)2704-2709
Number of pages6
JournalACS chemical biology
Issue number10
StatePublished - Oct 21 2022

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine


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