The Efficiency Advantage: Accelerating siRNA Manufacturing with Engineered Ligases

As RNA therapeutics expand beyond niche indications and move toward broader clinical use, the expectations placed on oligonucleotide manufacturing are evolving. Speed, scalability, and reproducibility in operations are central to competitive advantage.

One of the most critical steps in producing siRNA and other duplexed RNA constructs is enzymatic ligation: the joining of shorter RNA fragments into a full-length double-stranded RNA product. Yet this step is often a bottleneck, constrained by enzyme kinetics, reaction tolerance, and substrate limitations.

Recent advances in enzyme engineering have opened new opportunities to address these limitations. By tailoring ligases for manufacturability — not just reactivity — it’s now possible to improve both batch-level performance and overall process efficiency. This article explores the core dimensions of ligation efficiency in siRNA manufacturing and how engineered ligases are enabling faster, more scalable production.

Faster reaction kinetics: Compressing cycle time without compromising performance

In any ligation-driven RNA workflow, time-to-conversion is a key lever of efficiency. Slower ligation steps extend batch durations, increase enzyme consumption, and limit facility flexibility. For developers working under compressed timelines, these delays can accumulate, impacting everything from tech transfer windows to clinical readiness.

Engineered ligases can dramatically improve this picture. In comparative assessments using model siRNA duplexes, optimized ligases have demonstrated >95% conversion within one hour under standard manufacturing conditions. This is a marked improvement over typical wild-type enzymes, which may require two to four hours to reach similar endpoints — if they reach them at all.

Faster ligation enables shorter processing windows, more responsive production scheduling, and more efficient batch turnover — benefits that compound when working across multiple targets or iterative sequence updates.

High substrate loading: Bigger batches, fewer runs

For siRNA manufacturers, the ability to load high concentrations of substrate into a ligation reaction without compromising performance is a critical driver of batch efficiency. When enzymes struggle at higher loads, it forces production teams to split runs into smaller volumes, increasing the number of batches required to meet demand — along with the associated time, labor, and validation burden.

Engineered ligases offer a practical solution to this bottleneck. In performance evaluations, certain variants have shown high conversion rates at substrate concentrations up to 80 g/L — a significant leap from the 20–30 g/L ceiling typically observed with wild-type enzymes. This capability enables larger batch outputs, reduces the need for multiple runs, and supports more consistent downstream integration.

Importantly, this improvement in throughput doesn’t come at the expense of product quality. High-concentration ligations with optimized enzymes have maintained fidelity and duplex integrity, allowing for greater process intensification without introducing additional purification steps.

Robust performance under real-world conditions

Manufacturing rarely happens under perfect conditions. From scale-up variations to shifts in raw material quality, enzyme performance must hold up across a range of environments — beyond idealized lab parameters.

This is where process robustness becomes essential. Engineered ligases designed for manufacturing applications have demonstrated stable activity across broader temperature and pH windows compared to their wild-type counterparts. That stability allows greater flexibility in process design and makes it easier to transfer protocols across facilities or adapt them for different equipment platforms.

In addition, some ligase variants show tolerance to impurities present in crude RNA fragment pools. This can reduce the need for intensive pre-ligation purification steps, simplifying workflows and improving turnaround times — especially in fast-moving development settings.

By enabling enzymes to maintain performance under varied conditions, manufacturers gain more room to optimize around the broader process rather than constantly adapting the process to fit the enzyme.

Versatility and fit-for-purpose design

No two siRNA constructs are exactly alike, and neither are the conditions under which they’re manufactured. That’s why enzyme flexibility is such a critical component of ligation efficiency. From fragment length to backbone chemistry, developers need ligases that can accommodate diverse designs without extensive reengineering of the process.

Codexis has developed a library of engineered dsRNA ligases to support this need. These enzymes are screened and optimized to match a wide range of manufacturing scenarios, including:

• Compatibility with modified backbones such as 2′-OMe, 2′-F, and phosphorothioate linkages
• Support for variable fragment lengths and dual-nicked duplex assemblies
• Selectivity for properly annealed fragments, helping reduce misligation and downstream impurities

This adaptability enables more modular, design-forward workflows, where synthesis strategies can be built around the therapeutic construct rather than constrained by the tool.

From better steps to better systems

In isolation, each improvement in ligation efficiency — faster reactions, higher substrate tolerance, broader operating conditions, better fit-for-purpose design — contributes value. However, the true impact is seen when these gains accumulate across the full process.

Shorter ligation times translate to compressed batch timelines. Higher substrate loading increases per-run output and reduces the number of batches needed to meet demand. Robust enzymes that tolerate variable inputs reduce failure rates and ease process transfer between sites. Flexible enzyme selection enables developers to adapt workflows quickly as constructs evolve — without needing to revalidate from scratch.

For manufacturing teams balancing cost, speed, and quality at scale, these improvements don’t just enhance a single step — they change how production is planned, resourced, and executed.

At Codexis, our enzyme library and optimization support are designed to deliver on that broader promise. Whether enabling parallel fragment synthesis, streamlining ligation, or building toward continuous workflows, the goal is the same: to remove friction from RNA manufacturing, one optimized enzyme at a time.

Enzyme-enabled efficiency in action: Unlock the full white paper

Efficiency in siRNA manufacturing isn’t a single innovation but the result of many coordinated improvements across the process. Enzyme engineering plays a central role in that shift, especially as manufacturers look to scale duplex RNA production with speed, precision, and adaptability.

At Codexis, we have applied decades of experience in protein engineering to build a ligase library that meets these evolving demands — supporting faster development, higher batch output, and robust performance across real-world conditions.

Our latest white paper, Efficient siRNA Production Using Optimized dsRNA Ligases, explores the data behind these claims — including side-by-side comparisons of ligase performance, substrate titrations, and condition optimization. It’s essential reading for anyone advancing oligo manufacturing strategies in 2025 and beyond.

Download the white paper here: Improving siRNA Production Efficiency :: Codexis, Inc. (CDXS)

If you’re looking to streamline your RNA manufacturing workflows — or overcome bottlenecks in ligation, yield, or process stability — our team is here to help.