BioSpring Update September 2026 - Enzymatic ligation, CpG 7909 and insights from our Head of QC

Enzymatic ligation has become an increasingly relevant topic in oligonucleotide manufacturing. One obvious reason is the growing interest in longer and more complex RNA molecules. As sequences get longer, conventional solid-phase synthesis becomes more demanding, particularly when cumulative coupling losses lead to exponentially lower crude purity. But achieving new lengths is only part of the story.

Ligation-based approaches are also being explored for shorter therapeutic oligonucleotides, including ASOs and siRNA. Here, the rationale can be different. Instead of synthesizing a complete strand in one continuous process, shorter chemically synthesized fragments are produced separately and then joined enzymatically. Depending on the molecule and process design, this may create different options for scale-up and process efficiency or offer another way of dealing with certain impurities.

These potential advantages are highly process-specific, however. Fragment synthesis needs to deliver consistently high coupling efficiencies, while ligation and downstream processing must perform well enough to compete with an already highly optimized linear process. This is particularly relevant for siRNA, where linear manufacturing is well established, with substantial regulatory precedent worldwide, and remains the most straightforward route for many programs. Fragment ligation may nevertheless become particularly interesting in selected cases, including at very large manufacturing scales.

At BioSpring, linear solid-phase synthesis remains an established and important manufacturing platform, including long and chemically modified RNA. Our work with fragment ligation therefore does not start from the assumption that one technology should replace the other. Instead, it adds another option where the characteristics of a particular molecule or manufacturing process make a different approach worth considering.

Download our recent poster:From Linear Synthesis to Ligation - Comparative Insights Into Long Nucleic Acid Production

Where fragment ligation can make a difference

Our work to date has focused particularly on utilizing fragment ligation for long (>100 nt) and very long (>130 nt) RNAs, such as guide RNAs. For these molecules, the rationale for evaluating fragment ligation is straightforward. Every additional nucleotide adds another synthesis cycle. Even when individual coupling steps perform very well, small losses accumulate over a long sequence and can eventually affect overall yield and product quality. Purification can become increasingly demanding as well.

Fragment ligation changes that manufacturing route. The full RNA sequence is divided into shorter fragments, which are first produced individually by chemical synthesis. These fragments are then assembled enzymatically to generate the full-length product, for example, through splint-mediated ligation.

Even supposedly minor differences in coupling efficiency ("per-step-yield") of solid-phase synthesis have a dramatic impact on the purity of the crude product. 

The benefit depends heavily on the molecule.

In BioSpring’s studies with a 100 nt guide RNA, linear synthesis and fragment ligation delivered comparable performance. In another study with a 160 nt sgRNA, the picture was different: the ligation process resulted in higher product purity than linear synthesis.

This is also why we would avoid defining a simple sequence-length threshold at which one technology suddenly becomes preferable. Length certainly matters, but so do the way fragments are designed, the position of the ligation sites, secondary structure and chemical modifications. Enzyme compatibility can influence the outcome as well. The manufacturing strategy needs to account for these factors together.

Good ligation starts with good fragments

There is another important aspect that can easily get lost when enzymatic ligation is discussed as an alternative synthesis technology: the fragments still have to be made.

Basic principle of fragment ligation: Synthesis of shorter fragments and subsequent enzymatic ligation step (with or without DNA template).

Their quality has a direct influence on the subsequent ligation step. In our studies, higher-quality starting fragments were associated with better ligation efficiencies. Strong capabilities in chemical synthesis and purification therefore remain highly relevant even when the final molecule is assembled enzymatically.

This connection is important for how BioSpring approaches the technology. Fragment ligation builds on the knowledge gained from manufacturing long and complex oligonucleotides by established chemical synthesis. It adds another process step and another set of possibilities, but it does not remove the need to understand the underlying chemistry.

Moving from a successful reaction to a manufacturing process

Achieving an efficient ligation reaction at the development scale is one thing. Translating it into a process suitable for larger-scale manufacturing raises a different set of questions.

Individual fragment production has to remain consistent. The ligation reaction itself needs to perform reliably as scale increases, and downstream processing must handle the resulting product and impurity profile. Ultimately, what matters is whether the complete process can deliver the required product quality in a robust manufacturing setup.

BioSpring has built extensive experience in transferring linear guide RNA processes from development into larger-scale manufacturing, with scalability of both synthesis and purification considered from the outset. We have applied the same principles to our fragment ligation process, allowing scale-up requirements to be addressed early in development. Close collaboration between process development and manufacturing has supported the transfer into larger-scale production, including under GMP, while maintaining process performance and product quality.

The growing interest in enzymatic ligation is therefore broader than the question of how to make an exceptionally long RNA. Long guide RNAs provide a particularly clear use case and are an area in which BioSpring has already generated substantial process experience. At the same time, developments in fields such as siRNA suggest that fragment-based manufacturing concepts may become relevant for a wider range of therapeutic oligonucleotides.

Which route makes sense depends on the program

For some programs, established linear synthesis may remain the most appropriate approach. For others, fragment ligation may provide useful advantages as manufacturing requirements evolve. The decision also needs to consider factors beyond process performance alone. Compared with conventional linear synthesis, regulatory precedent for ligation-based manufacturing is still more limited, and intellectual property or freedom-to-operate considerations may also play a role. Having experience with both approaches gives BioSpring the opportunity to evaluate these factors alongside the actual process challenge rather than committing to a single manufacturing concept from the outset.