RNA molecules are not just simple copies of genetic instructions. They can carry chemical modifications that affect how stable they are, how they interact with cells, and how well they work as medicines. These modifications are especially important in therapeutic RNAs, where chemical changes can improve durability, delivery, and biological performance.

Researchers at Genentech examined how well nanopore direct RNA sequencing can detect a wide range of RNA modifications relevant to RNA therapeutics. Unlike many sequencing methods that convert RNA into DNA before analysis, nanopore direct RNA sequencing reads native RNA molecules directly, making it a promising approach for studying chemical modifications.

The researchers tested several types of modifications, including backbone, sugar, and base modifications. These included phosphorothioate linkages, 2′-O-methylation, 2′-fluoro, locked nucleic acid, 2′-O-methoxyethyl, N1-methylpseudouridine, 5-methylcytidine, 5-methoxyuridine, and 5-iodocytidine.

The results showed that some modifications were easier for nanopore sequencing to detect than others. Certain ribose modifications, N1-methylpseudouridine, and 5-methoxyuridine caused clear changes in sequencing errors and electrical current signals. Other modifications, such as phosphorothioate linkages, produced only subtle signal changes, making them more difficult to identify accurately.

The team also evaluated modification-aware basecalling models, which are computational tools designed to recognize specific RNA modifications. These tools could distinguish some modification types, but they still had limitations, including poor quantification accuracy and high local error rates, especially for 2′-O-methylation.

This work provides an important benchmark for the field. As RNA-based medicines continue to expand, researchers need reliable ways to measure and confirm RNA modifications. Nanopore direct RNA sequencing offers a powerful platform, but the results also show that more improvements are needed before it can fully support routine analysis of diverse therapeutic RNA modifications.

By clarifying both the strengths and limitations of the technology, the researchers provide a foundation for improving future RNA modification detection methods and advancing quality control for RNA therapeutics.