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

View ORCID ProfileWilliam Stephenson, Roham Razaghi, Steven Busan, Kevin M. Weeks, View ORCID ProfileWinston Timp, View ORCID ProfilePeter Smibert
doi: https://doi.org/10.1101/2020.05.31.126763
William Stephenson
1Technology Innovation Lab, New York Genome Center, New York, NY, USA
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  • For correspondence: physnano@gmail.com psmibert@nygenome.org
Roham Razaghi
2Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA
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Steven Busan
3Department of Chemistry, University of North Carolina, Chapel Hill, NC, USA
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Kevin M. Weeks
3Department of Chemistry, University of North Carolina, Chapel Hill, NC, USA
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Winston Timp
2Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA
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Peter Smibert
1Technology Innovation Lab, New York Genome Center, New York, NY, USA
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  • For correspondence: physnano@gmail.com psmibert@nygenome.org
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ABSTRACT

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.

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Competing Interest Statement

W.T. has two patents (8,748,091 and 8,394,584) licensed to ONT. W.S. and W.T. received reimbursement for travel, accommodation and conference fees to speak at events organized by ONT.

Footnotes

  • http://www.ncbi.nlm.nih.gov/bioproject/634693

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 01, 2020.
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Direct detection of RNA modifications and structure using single molecule nanopore sequencing
William Stephenson, Roham Razaghi, Steven Busan, Kevin M. Weeks, Winston Timp, Peter Smibert
bioRxiv 2020.05.31.126763; doi: https://doi.org/10.1101/2020.05.31.126763
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Direct detection of RNA modifications and structure using single molecule nanopore sequencing
William Stephenson, Roham Razaghi, Steven Busan, Kevin M. Weeks, Winston Timp, Peter Smibert
bioRxiv 2020.05.31.126763; doi: https://doi.org/10.1101/2020.05.31.126763

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