Systematic assessment of diverse RNA modifications using nanopore direct RNA sequencing

Area of Science:

• Molecular Biology
• Biotechnology
• Pharmacology

Background:

• Nanopore direct RNA sequencing advances transcriptomics but struggles with diverse therapeutic RNA modifications.
• Therapeutic RNAs utilize base, sugar, and backbone modifications for stability and efficacy.

Purpose of the Study:

• To systematically evaluate therapeutically relevant RNA modifications using direct RNA nanopore sequencing.
• To establish a performance baseline for nanopore technology in analyzing these modifications.

Main Methods:

• Analysis of phosphorothioate (PS), 2'-O-methylation (2'OMe), 2'-Fluoro (2'F), locked nucleic acid (LNA), 2'-O-(2'-methoxyethyl) (2'MOE), N1-methylpseudouridine (m1Ψ), 5-methylcytidine (m5C), 5-methoxyuridine (5moU), and 5-iodocytidine (5iodoC) modifications.
• Evaluation using basecall errors, raw current signals, and modification-aware basecalling models.
• Assessment of modification-aware basecallers for 2'OMe and m5C.

Main Results:

• Ribose modifications, m1Ψ, and 5moU increased error rates and altered current signals.
• 2'OMe and 2'MOE impacted dwell time; PS linkages caused minor current changes without errors.
• Modification-aware basecallers showed limitations in quantification accuracy and local error rates, especially for 2'OMe.

Conclusions:

• Direct RNA nanopore sequencing has variable capabilities for detecting therapeutic RNA modifications.
• The study clarifies current limitations, guiding future development for RNA therapeutics analysis.