RNA based therapeutics are rapidly expanding across modern medicine. Messenger RNA vaccines, gene editing systems, and RNA based immunotherapies all rely on carefully designed RNA molecules to work correctly. Because these molecules directly influence biological processes inside cells, scientists must carefully measure their quality and structure before they can be used in clinical applications.

Researchers at Genentech have developed a method that uses nanopore direct RNA sequencing to analyze several important RNA quality features at the same time. This approach allows scientists to study individual RNA molecules directly, providing detailed information about their sequence, structure, and integrity.

Nanopore direct RNA sequencing works by passing RNA molecules through tiny protein pores while measuring changes in electrical signals. These signals reveal the sequence of the RNA as well as structural features that can influence how the molecule functions. Because this technology reads native RNA molecules, it allows scientists to evaluate several critical quality attributes without converting RNA into DNA or using multiple separate tests.

The research team demonstrated that this approach can accurately determine the full length sequence of messenger RNA molecules and assess whether the RNA remains intact or has degraded during production. RNA integrity is important because damaged RNA molecules may produce incomplete or ineffective proteins.

One particularly important RNA feature is the presence of a chemical structure called the 5′ cap. This cap protects messenger RNA from degradation and helps cells recognize the RNA for protein production. The researchers developed a sequencing based method that directly measures how efficiently RNA molecules receive this protective cap.

The team also evaluated poly(A) tail length, another structural element that influences RNA stability and translation efficiency. The poly(A) tail is a stretch of adenine nucleotides at the end of messenger RNA molecules that helps regulate how long the RNA remains active inside cells.

In addition to messenger RNA, the method was used to analyze single guide RNA molecules used in CRISPR Cas9 genome editing. These short RNA molecules guide the Cas9 enzyme to specific DNA targets in the genome. By adding a specialized adapter, the researchers were able to sequence full length sgRNA molecules and confirm their identity.

The team compared their results with traditional analytical techniques such as chromatography and mass spectrometry. The sequencing based measurements closely matched results obtained from these established methods, demonstrating that nanopore sequencing can serve as a reliable analytical platform.

The researchers also examined how RNA quality affects genome editing performance. They found that when Cas9 messenger RNA molecules became degraded, the efficiency of gene knockout experiments decreased. This observation highlights the importance of monitoring RNA integrity during therapeutic development.

Together, these findings show that nanopore direct RNA sequencing can provide a unified approach for evaluating multiple RNA quality attributes in a single experiment. As RNA based medicines continue to expand, tools like this will help researchers ensure that therapeutic RNA molecules meet strict safety and performance standards.