Enzymatic DNA synthesis: Moving beyond limits

Dr Thomas Ybert, Co-founder and Chief Scientific Officer at DNA Script, describes how recent advancements in enzymatic DNA synthesis are unlocking previously inaccessible regions of the genome. Synthetic nucleic acids are foundational tools in biological research and therapeutic development, essential for applications ranging from gene synthesis to mRNA vaccine templates. As therapeutic ambitions grow, particularly in gene and cell therapies and RNA medicines, the demand increases for longer, more complex, and highly accurate nucleic acids. Traditional phosphoramidite chemical synthesis, the long-standing standard, struggles with challenging sequences (e.g., high GC content, repeats) and has inherent length and purity limitations for modern needs. These bottlenecks increasingly limit innovation. Enzymatic DNA synthesis (EDS) offers a powerful alternative, using biological catalysts in aqueous conditions to create the complex nucleic acids required for next-generation therapies. The scale and complexity of synthetic nucleic acids required by researchers have increased dramatically in recent years. Modern therapeutic strategies increasingly rely on sophisticated nucleic acid constructs that push the boundaries of traditional synthesis. Designing optimal mRNA vaccines often necessitates long DNA templates (many kilobases) incorporating intricate untranslated regions (UTRs). These UTRs frequently harbor GC-rich motifs or complex secondary structures crucial for mRNA stability and translational efficiency, but they represent significant hurdles for chemical synthesis. Similarly, antibody engineering frequently involves screening large variant libraries to identify candidates with improved affinity or novel functions. This process requires the efficient and accurate synthesis of lengthy gene fragments encoding diverse antibody formats, including complex structures like bispecific antibodies. Furthermore, the rapidly advancing field of adeno-associated virus (AAV)-based gene therapies relies heavily on precisely synthesised viral vector components, particularly the inverted terminal repeats (ITRs). The palindromic nature and propensity to form stable secondary structures make ITRs notoriously difficult to synthesise chemically with the high fidelity required for safe and effective gene delivery vectors. The inability to reliably access these complex sequences hampers innovation across these critical therapeutic areas. Limitations of traditional chemical synthesis Phosphoramidite chemistry faces challenges with today's long, complex sequences. Its multi-step cycle uses harsh, anhydrous conditions, and per-step coupling efficiencies below 99.5% cause exponential yield drops with increasing length. Synthesising sequences beyond ~200 bases yields low amounts of correct product, often dominated by deletion errors and truncations, requiring extensive purification that increases costs and turnaround times. Chemical synthesis is also sensitive to sequence composition; high GC content (>65%), hairpins, or repeats impede reactions, reducing yields and increasing errors, especially for elements like AAV ITRs. Harsh solvents and deprotection chemicals can damage DNA bases, compromising sequence integrity. The process also generates significant hazardous waste. These limitations lead to longer turnaround times, increased costs, failed synthesis attempts for ‘unmanufacturable’ sequences, and design constraints. Enzymatic DNA synthesis EDS uses engineered enzymes, such as terminal deoxynucleotidyl transferase (TdT) variants to construct DNA differently, under milder, aqueous conditions. Key features include: Template-Independent Enzymatic Addition: Enzymes sequentially add nucleotides without a template. Controlled Stepwise Synthesis: Reversible terminator nucleotides ensure single-base addition per cycle. Aqueous, Mild Conditions: Synthesis occurs in aqueous buffers near physiological pH and temperature, avoiding harsh chemicals. This enzymatic approach addresses many chemical synthesis limitations. Milder conditions reduce DNA damage like depurination. As the enzymatic addition process is not driven by the same hybridisation-dependent mechanisms as chemical coupling, it is generally less hindered by complex secondary structures or high GC content, allowing for higher fidelity synthesis of sequences that are challenging for phosphoramidite chemistry. The use of engineered, sometimes thermostable, TdT variants can further enhance synthesis efficiency and accuracy. From an environmental perspective, EDS drastically reduces the generation of hazardous chemical waste compared to traditional methods. EDS technology (Figure 1) combines two key capabilities: (i) the ability to produce high-quality standard-length oligos with high fidelity, and (ii) synthesise long, complex oligos that go beyond the limitations of chemical methods. This dual capability enables access to a range of sequences from routine synthesis of high-purity oligos up to 120 nt on benchtop systems, to high-quality synthesis of fragments of up to 500 nt through synthesis services. Internal benchmarking at DNA Script has shown that sequences often considered 'hard' or too complex (e.g., 1.5 kb–7 kb with secondary structures or high GC content) and often declined by chemical providers can be successfully synthesised and assembled using EDS oligonucleotides. These results highlight the potential of EDS to overcome common barriers in DNA synthesis. Therapeutic frontiers and complex gene assembly The capacity of EDS technology to produce long, complex DNA with high fidelity is directly impacting therapeutic research and simplifying complex gene assembly workflows. EDS facilitates the production of long DNA templates required for generating complex mRNA constructs. This allows researchers greater freedom to optimise 5' and 3' UTRs containing difficult motifs or to incorporate precisely defined long or segmented Poly(A) tails, potentially improving mRNA stability, translation efficiency, and overall vaccine or therapeutic efficacy. In the quest for better antibodies, EDS can streamline the generation of diverse DNA fragments needed to build libraries encoding antibody variants or complex formats like bispecific or multispecific antibodies. Faster access to these DNA building blocks can significantly accelerate the discovery and development pipelines for antibody-based therapeutics. EDS also provides a more robust and reliable pathway for synthesising high-fidelity ITR sequences, overcoming a major bottleneck in the field of AAV-based therapies. Accurate and efficient ITR synthesis is critical for producing safe and potent AAV vectors, and improvements here can accelerate AAV vector development, process optimisation, and manufacturing. Beyond synthesising specific components, EDS significantly aids the assembly of complete genes or large functional DNA fragments that are difficult or impossible to construct using traditional oligos. By using longer, high-fidelity EDS-synthesised oligos (e.g., 120-mers or longer), complex gene assembly can be achieved more rapidly and accurately. For instance, researchers have reported assembling kilobase-scale genes in just a few days using EDS-generated oligos in standard assembly methods like Polymerase Cycling Assembly (PCA) or Gibson assembly. The use of longer starting fragments (e.g., 120-mers from EDS vs. ~60-mers from chemical synthesis) can reduce the number of fragments needed for assembly, simplify the process, and improving the fidelity of the final construct. Furthermore, EDS offers valuable solutions when target sequences are incompatible with standard assembly workflows, particularly those relying on error correction methods that struggle with highly repetitive regions. High-quality, long EDS oligos can be used directly in overlap-based assembly methods such as Gibson assembly, bypassing problematic enzymatic steps or the need for extensive sub-cloning. Studies have demonstrated the efficient direct assembly of challenging sequences using long EDS oligos, including fragments with very high GC content (~80%) and significant repeat structures, fragments with tandem repeats and poly-G stretches, long Poly(A) tail sequences (>150 nt), and complex multi-domain fragments like CAR-T scFvs assembled from multiple oligos up to 450 nt each, achieving respectable percentages of perfect clones directly from assembly. The future of EDS Traditional chemical synthesis faces undeniable limitations when confronted with the most complex sequences required by modern research. EDS is rapidly evolving, with ongoing improvements in synthesis speed, achievable length, sequence fidelity, and overall cost-effectiveness. These advancements position EDS as an increasingly crucial technology for overcoming synthesis bottlenecks that currently impede discovery and development. By leveraging the precision of enzymes operating under mild, aqueous conditions, EDS gives researchers access to challenging DNA sequences needed to advance studies of gene regulation, assemble difficult genes for synthetic biology applications, engineer novel protein and RNA therapeutics, and reshape the landscape of drug discovery. From DDW Volume 26 – Issue 3, Summer 2025 Read the digital issue About the author: Dr Thomas Ybert co-founded DNA Script in 2014 and, as CSO, leads the company's research and innovation efforts. Ybert is the main inventor of DNA Script's Enzymatic DNA Synthesis (EDS) technology. Holding a PhD in molecular biology and yeast genetics from Ecole Polytechnique, his background includes R&D roles at Sanofi and Total/Amyris.