Main menu

Direct detection of RNA modifications and structure using single molecule nanopore sequencing


Many methods exist to detect RNA modifications by short-read sequencing, relying on either antibody enrichment of transcripts bearing modified bases or mutational profiling approaches which require conversion to cDNA. Endogenous modifications are present on several major classes of RNA including tRNA, rRNA and mRNA and can modulate diverse biological processes such as genetic recoding, mRNA export and RNA folding. In addition, exogenous modifications can be introduced to RNA molecules to reveal RNA structure and dynamics. Limitations on read length and library size inherent in short-read-based methods dissociate modifications from their native context, preventing single molecule analysis and modification phasing.

Here we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications over long sequence distance at the single molecule level.

We demonstrate comprehensive detection of endogenous modifications in E. coli and S. cerevisiae ribosomal RNA (rRNA) using current signal deviations. Notably 2’-O-methyl (Nm) modifications generated a discernible shift in current signal and event level dwell times. We show that dwell times are mediated by the RNA motor protein which sits atop the nanopore. Further, we characterize a recently described small adduct-generating 2’-O-acylation reagent, acetylimidazole (AcIm) for exogenously labeling flexible nucleotides in RNA. Finally, we demonstrate the utility of AcIm for single molecule RNA structural probing using nanopore sequencing.

Authors: William Stephenson, Roham Razaghi, Steven Busan, Kevin M. Weeks, Winston Timp, Peter Smibert

入门指南

购买 MinION 启动包 Nanopore 商城 测序服务提供商 全球代理商

纳米孔技术

订阅 Nanopore 更新 资源库及发表刊物 什么是 Nanopore 社区

关于 Oxford Nanopore

新闻 公司历程 可持续发展 领导团队 媒体资源和联系方式 投资者 合作者 在 Oxford Nanopore 工作 职位空缺 商业信息 BSI 27001 accreditationBSI 90001 accreditationBSI mark of trust
Chinese flag