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

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• RNA modifications regulate crucial biological processes like genetic recoding and mRNA export.
• Existing short-read sequencing methods limit analysis of RNA modifications in their native context.
• Single-molecule analysis of RNA modifications is essential for understanding their functional roles.

Purpose of the Study:

• To demonstrate direct RNA nanopore sequencing for detecting RNA modifications at the single-molecule level.
• To analyze endogenous and exogenous RNA modifications on long RNA molecules.
• To investigate RNA structure and dynamics using chemical probing with nanopore sequencing.

Main Methods:

• Direct RNA nanopore sequencing of long RNA molecules.
• Detection of endogenous 2'-O-methyl and base modifications in ribosomal RNAs from *E. coli* and *S. cerevisiae*.
• Utilizing a helicase motor protein to facilitate detection of modifications via shifts in current signal and dwell times.
• Employing the SHAPE reagent acetylimidazole for single-molecule RNA structure probing.

Main Results:

• Successfully detected endogenous RNA modifications on long RNAs at the single-molecule level using nanopore sequencing.
• Identified specific shifts in current signal and dwell times corresponding to 2'-O-methyl and base modifications in ribosomal RNAs.
• Demonstrated the capability to probe RNA structure using chemical reagents and direct nanopore sequencing readout.

Conclusions:

• Direct RNA nanopore sequencing is a powerful tool for single-molecule analysis of RNA modifications.
• This technique overcomes limitations of short-read sequencing, preserving the native context of modifications.
• The method provides new avenues for studying RNA structure, dynamics, and the functional impact of modifications.