UAlbany Researchers Advance New Technique for Direct RNA Sequencing

ALBANY, N.Y. (July 6, 2026) — RNA and DNA are molecular cousins. Both carry biological instructions and both are built from four basic building blocks. They also have their differences. One of these is our ability to “read” the full biological message that they carry. Science has been able to accomplish this for DNA, but RNA’s full message is still being discovered.

Part of what makes RNA more complicated to “read” is the fact that RNA molecules contain more than 170 modifications which cells use as a kind of “shorthand” for controlling gene expression, regulating proteins and fine-tuning biological processes. Current RNA sequencing technologies, which read sequences indirectly through color signals or electrical currents, excel at reading the four standard bases but are largely blind to modifications.

This leaves a critical gap: We lack a reliable way to determine RNA’s “true” sequence, including all of its chemical modifications. That limitation affects both basic research and the safety of RNA-based medicines.

University at Albany’s Shenglong Zhang, associate professor in the Department of Chemistry and the RNA Institute, is working to address this gap. His team recently published a new article in the journal Nucleic Acids Research, which details their latest technological advances that get us closer to being able to “read” RNA’s true sequence, including its primary building blocks, plus modifications implicated in disease. 

We caught up with Zhang, who also serves as founder and president of spinoff company DirectSeq Biosciences, Inc., to learn more about his team’s latest research and what it means for our ability to understand RNA diseases and develop RNA-based pharmaceuticals.

What did you set out to do?  

We are developing a fundamentally different approach to reading RNA sequences which uses a technique called Next Generation Mass Spectrometry sequencing (NGMS-Seq), to directly sequence an RNA strand using mass as the readout. Every RNA nucleotide building block — modified or not — has a unique mass, making mass spectrometry a natural fit for reading true, complete RNA sequences.

Our latest innovation builds on an earlier technique called the Sanger sequencing ladder model. It creates similar ladders for RNA through a highly controlled acid hydrolysis that cleaves each RNA molecule once. Each resultant fragment forms a rung of the sequencing ladder, and the mass difference between successive ladder fragments reflects the exact mass of the RNA nucleotide at that position, making it possible to read the full-length RNA sequence, including its modifications.

In our new paper, we lay a mathematical foundation of NGMS-Seq and report a major advance: adding signal intensity of ladder fragments as a third dimension alongside mass and retention time that were measured together by our liquid chromatography-mass spectrometry (LC-MS) instrument. This enables a fast, direct sequencing of not only purified RNA, but also of complex mixtures containing multiple RNA molecules, without any prior sequence input.

What did you find?

The key discovery here is that ladder fragment intensity reflects how much of each parent RNA was present — a relationship that allows us to computationally separate and independently sequence multiple RNA species present together in the same sample.

Applied to small therapeutic RNAs, including microRNAs (miRNAs) and CRISPR guide RNAs (sgRNAs) containing both standard and modified bases, the platform accurately determined complete, true sequences. In one case when sequencing a 100-nt CRISPR guide RNA, we discovered the “pure” sample was actually only 71.8% the intended product. The rest? A truncated impurity from a synthesis failure, plus minor isomers carrying unintended methylations — at the exact same mass as the intended modifications invisible to conventional sequencing methods but with potentially unintended risks.

More importantly, by directly sequencing heterogeneous RNAs without prior sequence input, 3D NGMS-Seq addresses key limitations of current RNA analysis, providing a powerful tool for small RNA drug development, quality control and regulatory validation.

How do your latest findings move the needle on our ability to ‘read’ RNA and understand its modifications?

This work represents a significant step toward true, comprehensive RNA sequencing with all modifications. The implications are significant in two directions.

First, for the RNA therapeutics industry: mRNA, siRNA and sgRNA drugs are now treating previously untreatable diseases, which is a major accomplishment for the field. Yet, variation in these drugs’ consistency across batches is a poorly understood concern. Many such events may stem from sequence impurities in synthetic RNA preparations — impurities that current methods cannot reliably detect. Our platform provides, for the first time, a way to not only detect these impurities, but pinpoint exactly where and how much they occur, which is critical because the same modification can be harmless in one position and harmful in another.

Second, for the emerging Human RNome Project — an international effort to map the complete RNA content of human cells across tissues, disease states and populations, including all modification types — our method offers a tool that existing technologies simply cannot provide: direct, accurate RNA sequencing across diverse biological samples, helping to close a longstanding gap in our understanding of RNA modifications or the “epitranscriptome” codes.

In short, our study introduces a direct, three-dimensional way to read RNA as it truly exists — capturing both standard and modified bases, even in complex mixtures — while bringing us closer to obtaining complete and reliable RNA sequence information for applications in science and medicine.