Toward consolidated RNA quality analysis with direct RNA sequencing

As RNA-based therapeutics continue to expand across vaccines, immunotherapies, and gene-editing approaches for cell and gene therapy development, regulatory expectations for RNA quality characterisation are evolving alongside them. Recent guidance from health authorities has placed increasing emphasis on comprehensive assessment of critical quality attributes (CQAs) such as sequence identity, transcript integrity, 5′ capping efficiency, poly(A) tail length, and functionality to help ensure product quality, consistency, safety, and efficacy^1,2.

However, these quality attributes are often measured using multiple orthogonal analytical methods. Sanger sequencing may be used for sequence verification, capillary gel electrophoresis for RNA integrity, and chromatography or mass spectrometry-based methods for structural features such as 5’ caps and poly(A) tail length. While valuable, these approaches can require multiple instruments, extensive sample preparation, specialist workflows, and repeated sample handling³, increasing turnaround time and creating more opportunities for variability and error.

Chatla et al. evaluated Oxford Nanopore direct RNA sequencing as a unified, single-molecule analytical approach for characterising messenger RNA (mRNA) and single-guide RNA (sgRNA) quality⁴ — both essential components of the CRISPR-Cas9 gene-editing system. Unlike workflows that rely on reverse transcription and PCR, direct RNA sequencing analyses native RNA molecules, reducing sample manipulation while retaining molecule-level resolution. Their work demonstrated how nanopore sequencing can consolidate analyses traditionally performed using Sanger sequencing, capillary gel electrophoresis, and chromatography or mass spectrometry-based methods into a single workflow. By combining full-length sequence information with measurements of RNA structure, integrity, purity, and stability, the approach generated results that are directly relevant to RNA therapeutic development and manufacturing.

Moving beyond fragmented RNA quality workflows

To assess direct RNA sequencing for mRNA characterisation, the team analysed two in vitro transcribed Cas9 mRNA constructs with different structural features. One construct contained a templated-encoded poly(A) tail and 3’ untranslated region (UTR), whereas the second had a shorter template-encoded poly(A) tail that was enzymatically extended and no 3’ UTR. The researchers prepared the samples using the Direct RNA Sequencing Kit and sequenced the resulting libraries on MinION RNA Flow Cells using a GridION device.

The team first investigated sequence confirmation, comparing direct RNA sequencing with Sanger sequencing using the Cas9 construct with a 3’ UTR. They found that Sanger sequencing successfully verified the coding region of the transcript, producing a consensus sequence with 100% identity to the expected reference.

This difference is particularly important for teams seeking complete transcript visibility. As each nanopore read corresponds to an individual RNA molecule, the approach preserved information across the full transcript architecture, including non-coding regions. The authors highlighted: ‘Unlike Sanger sequencing, the [direct RNA sequencing] approach was able to capture additional transcript features, including the 3’ UTR and poly(A) tail, offering a more comprehensive view of mRNA structure and integrity’.

By reducing dependence on reverse transcription, primer design, amplification, and fragmented sequence assembly, nanopore sequencing offers a more direct route to transcript-level characterisation than Sanger sequencing. For organisations working towards scalable RNA therapeutic workflows, this has important practical implications: fewer individual steps and a reduced risk of missing critical information.

Capturing multiple mRNA quality attributes with one platform

Beyond sequence confirmation, Chatla et al. showed that direct RNA sequencing with nanopore technology provides quantitative insight into several mRNA structural features.

For poly(A) tail analysis, the researchers compared nanopore sequencing against chromatography and mass spectrometry-based methods. All three methods consistently detected the full template-encoded poly(A) tail for the first construct.

The team also evaluated 5’ capping efficiency, demonstrating a quantitative sequencing-based approach for distinguishing capped and uncapped mRNA molecules. Using a Cas9 mRNA construct and a commercially available eGFP mRNA, the researchers selectively labelled uncapped mRNA molecules with a synthetic RNA barcode. They were able to differentiate between capped and uncapped populations through nanopore sequencing, and the results showed strong agreement with established analytical methods, while also providing a molecule-level view of capping heterogeneity.

The same workflow enabled the team to assess mRNA integrity. Using thermally stressed samples to simulate degradation, the researchers showed that direct RNA sequencing could detect time-dependent fragmentation and a reduction in full-length transcripts. This validated nanopore sequencing as a stability-indicating method for mRNA therapeutic analysis.

Linking RNA integrity to biological performance

The researchers next explored whether nanopore-derived quality metrics could predict functional outcomes by performing in vitro knockout assays in human T cells using fresh and thermally stressed Cas9 mRNA samples. Fresh mRNA samples consistently achieved more than 95% knockout efficiency. However, the team observed that as mRNA degradation increased, functional activity declined. Their result indicates that direct RNA sequencing can deliver functionally predictive metrics of mRNA integrity, which are essential for quality assessment of gene-editing components.

Extending direct RNA sequencing to sgRNA characterisation

Chatla et al. also expanded nanopore direct RNA sequencing to include sgRNAs, the molecules that guide CRISPR-Cas9 systems to their genomic targets. Unlike mRNAs, sgRNAs do not contain a poly(A) tail, which is normally required for library preparation with the standard Direct RNA Sequencing Kit.

To address this, the team developed a custom workflow to adapt the standard kit. This meant they could directly sequence non-polyadenylated sgRNAs. This work shows how the same platform can support both mRNA and sgRNA characterisation for gene-editing applications.

Understanding how sgRNA impurities affect editing outcomes

In a related study, the researchers investigated whether sgRNA quality attributes could be linked directly to gene-editing performance. Combining chromatography, mass spectrometry, and direct RNA sequencing, they characterised different classes of sgRNA impurities and evaluated their impact on editing outcomes⁵.

Nanopore sequencing revealed elevated deletion rates in the target-specific region (the first 20 nucleotides) of certain sgRNA elution fractions, indicating the presence of 5′-truncated molecules. Functional testing showed that this class of impurity reduced knockout efficiency in a dose-dependent manner, with the highest impurity levels (a 30% spike in) decreasing editing performance by approximately 15%.

Together, these findings reinforce an important principle for RNA therapeutic development: quality attributes are not simply analytical measurements. By connecting RNA quality directly to functional performance, nanopore sequencing can provide actionable insight that supports the development of more consistent and effective gene-editing therapies.

Supporting future-ready RNA quality workflows

As RNA therapeutics move through development and manufacturing, consolidated quality analysis could help reduce complexity while providing more comprehensive insight into product quality. As Chatla et al. concluded, nanopore direct RNA sequencing is a ‘powerful, versatile platform for RNA characterization, combining full-length sequencing, mRNA capping, degradation detection, poly(A) tail profiling, and high-throughput sample analysis in a single workflow’ and is a ‘strong candidate for GMP-compliant quality control’⁴.

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