Direct RNA Sequencing Supports Novel Discoveries in RNA Biology

Years of experiments and databases filled with RNA-seq results belie the simple reality that, until fairly recently, it was impossible to analyze RNA directly. The RNA-seq studies performed with conventional sequencing platforms are based entirely on cDNA, an imperfect proxy for RNA that requires scientists to infer knowledge about the original RNA molecule rather than observe it directly.

It is understandable that the scientific community accepted this compromise. Single-stranded RNA is dynamic and highly structured, folding back on itself in ways that make it impossible for the polymerases used in most sequencing technologies to sequence an entire transcript. Repetitive sequences, hairpin structures, modifications, and vulnerability to degradation—RNA harbors a number of challenges that collectively prevent direct sequencing.

While cDNA-seq has been a necessary and important approximation for learning more about RNA, it is past time for scientists to achieve direct access. That’s not just the general sentiment—it’s the upshot of a report issued this year by the National Academies of Sciences, Engineering, and Medicine (NASEM) as part of a call for a 15-year initiative that would ultimately allow researchers to sequence RNA molecules with all of their modifications intact.1 The report suggests that this program could be modeled on the Human Genome Project, with similar efforts to build out RNA-seq capacity.

RNA holds valuable information that can help scientists learn more about basic biology as well as healthy states versus disease states—information that could be used to develop new RNA-based therapies and vaccines. In an announcement about the NASEM report, Victor J. Dzau, MD, president of the National Academy of Medicine, said, “Understanding RNA modifications and harnessing this knowledge holds immense potential—not only for human health and medicine but also for shaping all living systems and the products and technologies stemming from them.”2

Even as new efforts are inspired by the NASEM report, some scientists are already making progress in RNA analysis by deploying nanopore-based sensing technologies. The same nanopore platforms used for sequencing DNA can also be used with RNA, enabling direct analysis of the molecule without the need for a cDNA conversion step. Initial studies based on this approach have already led to novel findings in RNA modifications and noncoding RNAs, as well as assuring quality for RNA-based therapies.

RNA modifications

Like the epigenetic changes seen on DNA, RNA modifications are essential to the molecule’s function. They are often stripped away in the cDNA conversion process, but direct RNA analysis has made it possible to resolve these modifications in detail for the first time.

Recent studies demonstrate the power of characterizing RNA modifications. In a study at the University of Colorado School of Medicine, scientists analyzed post-transcriptional modifications in transfer RNAs from six diverse species consisting of five eukaryotes and one prokaryote.3 Using high-yield libraries for sequencing, the scientists generated extensive coverage of the molecules and identified 43 separate RNA modifications. These changes included mitochondrial-specific modification in a eukaryotic species and insight into bacterial tRNA modifications that occurred during infection by a bacteriophage. Overall, the team reported that the work establishes a strong foundation for interrogating tRNAs with direct RNA analysis.

In a study of the effects of spaceflight on human physiology, researchers from Weill Cornell Medicine and other institutions performed direct RNA-seq on samples collected from four astronauts at seven different time points, starting prior to a three-day flight and ending after a post-flight recovery period.4 The transcriptome results generated via RNA-seq shed light on several changes to genetic pathways influenced by spaceflight, including alterations to stress and the immune response. RNA modifications were also found to be changed: an analysis of modifications across isoforms indicate “a significant spike in m6A levels immediately post-flight,” the scientists reported. “These data and results represent the first longitudinal long-read RNA profiles and RNA modification maps for each gene for astronauts, improving our understanding of the human transcriptome’s dynamic response to spaceflight.”

Noncoding RNAs

Noncoding RNAs, especially long ones, have been particularly difficult to analyze with any technology because they so often include highly repetitive sequences. With direct RNA analysis, though, scientists are finally tapping into some of the secrets of noncoding RNA.

For instance, a study from scientists at the University of York and St. James’s University Hospital in the United Kingdom used direct RNA-seq to analyze resected samples of clear cell renal cell carcinoma, a common type of kidney cancer.5 The discovery cohort of 12 samples and a validation cohort of 20 samples originally came from patients who went on to have a variety of outcomes from their cancer. Across the board, the study revealed a wealth of data about RNAs that had never been seen before, including more than 10,000 novel transcripts. Among the more intriguing discoveries was a novel noncoding gene that was overexpressed in patients who eventually relapsed, but not in other patients.

University of Oklahoma researchers studied the human immune response to influenza by analyzing RNA in bronchial epithelial cells. They focused on noncoding RNA, both polyadenylated and nonpolyadenylated, tracking changes in expression and modifications in response to infection.6 Interestingly, two long intergenic noncoding RNAs (lincRNAs) that had recently been associated with immune response were flagged in this project; both lincRNAs became highly methylated after exposure to the flu virus. Shorter noncoding RNAs were also altered after infection, highlighting a number of prospects that will be important for future investigations into the immune response to viruses.

Quality control for RNA therapies

While most scientists are turning to direct RNA-seq to help advance our understanding of RNA biology in health and disease, the same approach has also been used in applied research. With the explosion of interest in RNA-based therapies that followed the success of the COVID-19 mRNA vaccines, some scientists have been incorporating direct RNA analysis for quality control and assurance processes for RNA-based therapies and vaccines.

At the University of Queensland and other institutes in Australia, for example, scientists reported an mRNA vaccine quality analysis protocol based on nanopore sequencing called VAX-seq.7 Typical quality control methods used for this new class of therapies are plagued by high costs and long timelines, but according to these scientists, direct RNA-seq can be used to streamline the process.

“VAX-seq can comprehensively measure key mRNA vaccine quality attributes, including sequence, length, integrity, and purity,” the researchers noted. “Given these advantages, we anticipate that RNA-seq methods, such as VAX-seq, will become central to the development and manufacture of mRNA drugs.”

As more biotechnology and pharmaceutical companies race to develop RNA-based therapies, protocols such as VAX-seq will be important for ensuring that each treatment contains the desired RNA content without any contaminants.

Looking ahead

Novel findings based on direct RNA analysis are routinely being reported across species, disease states, applications, and more. At least some of these results will advance our understanding of biology, and others will ultimately make it possible to deliver better treatments to patients. As the underlying technology continues to improve, these explorations of RNA will get even deeper and more detailed.

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