A New Approach To RNA Synthesis And Purification: Rethinking A Persistent Bottleneck

By Maksim Royzen, Ph.D., associate professor, University at Albany

The synthesis and purification of RNA oligonucleotides remain foundational challenges in the development of RNA-based therapeutics. While advances in sequence design, delivery systems, and chemical modification have accelerated the field, upstream manufacturing constraints continue to limit scalability, cost efficiency, and product quality.

One of the most persistent bottlenecks lies in purification. Traditional methods, particularly high-performance liquid chromatography (HPLC) and gel-based purification, are effective but resource-intensive, time-consuming, and difficult to scale. These approaches often become the limiting step in producing long or highly modified RNA sequences, especially as demand grows for increasingly complex constructs.

A recent study introduces a different paradigm: instead of improving chromatography, it seeks to eliminate the need for it altogether.

Photocleavable Supports As A Platform Innovation

At the center of this approach is a coumarin-based photolabile solid support designed for RNA synthesis. This support acts as an anchor during solid-phase synthesis, enabling the RNA strand to be assembled step by step. The key innovation lies in how the RNA is released.

Rather than relying on chemical cleavage followed by chromatographic purification, the system uses light to trigger release. Upon irradiation, the coumarin linker undergoes a photochemical reaction that cleanly detaches the RNA from the solid support.

This seemingly simple shift has important implications. Because truncated or failure sequences remain bound to the support or are otherwise separable, the released RNA is enriched for full-length product without requiring traditional purification workflows.

In effect, purification is partially built into the synthesis process itself.

Eliminating Chromatography: Why It Matters

Chromatography has long been the gold standard for oligonucleotide purification, but it introduces several challenges:

• Scalability constraints: Large-scale chromatography requires significant infrastructure and solvent use.
• Time and cost: Multiple purification steps increase manufacturing timelines and expenses.
• Material loss: Each step can reduce yield, particularly for longer RNA constructs.

By contrast, a nonchromatographic approach offers a more streamlined pathway. If purity can be achieved during synthesis and release, downstream processing becomes significantly simpler.

This is particularly relevant as RNA therapeutics expand beyond short oligonucleotides into longer, more complex molecules. As sequence length increases, so does the difficulty of separating full-length product from closely related impurities. A method that reduces reliance on separation altogether could represent a meaningful shift in manufacturing strategy.

Mechanistic Design: Coumarin As A Functional Linker

The choice of coumarin is not incidental. Coumarin-based systems are well known for their photochemical properties, including efficient and controllable cleavage under specific wavelengths of light.

In this context, the coumarin linker serves multiple roles:

• Stable during synthesis: It withstands the chemical conditions required for RNA assembly.
• Selective cleavage: It can be activated by light without damaging the RNA product.
• Compatibility: It integrates into existing solid-phase synthesis workflows.

This balance is critical. Any alternative to standard supports must not disrupt the underlying chemistry of RNA synthesis, which is already highly optimized.

The study demonstrates that the coumarin-based support meets these criteria, enabling efficient synthesis while introducing a new release mechanism that doubles as a purification step.

Performance Across RNA Lengths And Applications

A key question for any new synthesis method is whether it generalizes across different RNA constructs.

The system was evaluated across multiple oligonucleotide lengths and sequences, including those relevant to gene editing applications. The results showed that high-purity RNA could be obtained without traditional chromatographic purification, even for longer sequences that typically pose greater challenges.

Importantly, the resulting RNA was not only structurally intact but also functionally active. In downstream assays, including applications involving CRISPR systems, the RNA performed comparably to conventionally purified material.

This functional validation is essential. For RNA therapeutics, purity is not just a chemical metric — it directly impacts biological activity, safety, and regulatory acceptance.

Integration With Gene Editing Workflows

One of the more compelling aspects of this approach is its compatibility with gene editing systems such as CRISPR.

Guide RNAs (gRNAs) used in CRISPR applications must meet stringent quality requirements. Impurities or truncated sequences can reduce editing efficiency or introduce off-target effects. At the same time, demand for these molecules is increasing rapidly, both in research and therapeutic contexts.

By enabling the synthesis of high-quality RNA without chromatography, this method could streamline the production of gRNAs and related molecules. The ability to generate functional RNA directly from synthesis and light-triggered release simplifies workflows and may reduce barriers to scale.

This is particularly relevant for applications requiring rapid iteration, such as screening or personalized therapies.

Broader Implications For RNA Manufacturing

The significance of this work extends beyond a single chemical innovation. It reflects a broader shift in how the field is thinking about RNA manufacturing.

Traditionally, synthesis and purification are treated as separate stages. First, the molecule is built; then, it is cleaned. This separation introduces inefficiencies, especially as molecules become more complex.

The approach described here blurs that boundary. By embedding purification into the synthesis and release process, it reduces the need for downstream intervention. This aligns with a more integrated view of manufacturing, where each step is designed not only for its primary function but also for its impact on overall process efficiency.

If broadly adopted, such strategies could:

• reduce manufacturing costs
• improve scalability
• increase throughput for complex RNA constructs
• simplify regulatory validation by reducing process variability.

These advantages become increasingly important as RNA therapies move from niche applications to larger patient populations.

Limitations And Considerations

Despite its promise, this approach is not without challenges.

First, the use of light as a trigger introduces new variables. Ensuring uniform irradiation at scale, particularly in large or opaque systems, may require additional engineering. The wavelength and intensity of light must be carefully controlled to achieve efficient cleavage without damaging the RNA.

Second, while the method reduces reliance on chromatography, it may not eliminate the need for all downstream processing. Depending on the application, additional steps may still be required to meet regulatory standards, particularly for therapeutic use.

Third, integration into existing manufacturing pipelines will require validation. Pharmaceutical production environments are highly standardized, and new methods must demonstrate not only performance but also reproducibility, robustness, and compatibility with regulatory expectations.

These considerations do not diminish the value of the approach, but they highlight the work required to translate it from laboratory innovation to industrial practice.

A Shift Toward Process-Level Innovation

What makes this work particularly notable is its focus on process rather than product.

Much of the innovation in RNA therapeutics has centered on improving the molecules themselves: optimizing sequences, enhancing stability, or refining delivery systems. These advances are critical, but they must ultimately be matched by equally sophisticated manufacturing solutions.

Process-level innovations, like the one described here, address a different but equally important question: How do we make these molecules efficiently, consistently, and at scale?

In many ways, the future of RNA therapeutics will depend as much on manufacturing as on biology. Breakthroughs in delivery or design will have limited impact if they cannot be translated into scalable, cost-effective production.

Conclusion

The development of a coumarin-based photolabile solid support for RNA synthesis represents a meaningful step toward rethinking how RNA is produced and purified.

By enabling light-triggered release and reducing reliance on chromatography, this approach simplifies a traditionally complex workflow. It demonstrates that purification does not have to be a separate resource-intensive step but can instead be integrated into the synthesis process itself.

While further work is needed to scale and validate the method, the underlying concept is compelling. As RNA therapeutics continue to expand in scope and complexity, innovations that streamline manufacturing will play an increasingly central role.

In that sense, this is more than a new tool. It is an example of how reimagining the fundamentals of process design can unlock new possibilities across the RNA landscape.

To read the full article: Coumarin-Based Photolabile Solid Support Facilitates Nonchromatographic Purification of RNA Oligonucleotides.

About The Author

Maxsim “Max” Royzen, Ph.D., originally from the Soviet Republic of Moldova, received his bachelor’s degree in chemistry from Dartmouth College, where he conducted undergraduate research with Prof. Russell Hughes. Royzen earned his Ph.D. in physical organic chemistry in 2006 under the supervision of Prof. James Canary, where he investigated fluorescent properties of transition metal binding compounds. After a two-year stint in industry, Royzen joined the laboratory of Prof. Stephen Lippard, as a Ruth L. Kirschstein NIH postdoctoral fellow. In 2013, he joined the faculty in the Department of Chemistry at the University at Albany as an assistant professor. His research interests are thematically based on different aspects of molecular recognition at the interface of bio-organic and bioinorganic chemistry. Much of his lab focuses on developing new applications of bio-orthogonal chemistry that include drug delivery, RNA synthesis, and the investigation of RNA-protein interactions.