Enzymatic oligonucleotide synthesis (EOS) is a new approach to DNA synthesis, based on the use of enzymes such as DNA polymerases, reverse transcriptases and terminal deoxynucleotidyl transferases (TdT) to synthesize oligonucleotides under aqueous conditions relevant to biology. In contrast to the commonly used phosphoramidite approach to DNA synthesis, which requires the use of harsh, organic solvent conditions and which generates large amounts of toxic waste, the intrinsic selectivity and aqueous, mild nature of enzymes allows them to be used to incorporate nucleotides one at a time in highly efficient fashion. EOS has the potential to greatly impact synthetic biology, personalized medicine, gene editing and high-throughput genomics.
Figure 1. Pros and cons of chemical automated synthesis and enzymatic synthesis of oligonucleotides[1].
Alfa Chemistry recognizes the transformative potential of EOS and actively supports the development of this field by offering custom oligonucleotide synthesis services, specialized enzyme systems, and a catalog of nucleotide analogs and solid-phase supports designed for enzymatic workflows.
Terminal deoxynucleotidyl transferase (TdT) is the cornerstone of template-independent EOS due to its ability to add deoxynucleotide triphosphates (dNTPs) directly to the 3'-hydroxyl end of a single-stranded DNA without requiring a guiding template. Since TdT's natural function involves random incorporation, researchers have developed chemical blocking strategies to ensure controlled, base-by-base addition.
One prominent approach involves using 3'-O-caged dNTPs, which bear removable blocking groups. In a two-step cycle—nucleotide addition followed by deprotection—TdT can be directed to sequentially extend immobilized primers. However, this method is limited by the enzyme's steric constraints, as TdT struggles to accommodate bulky modifications at the 3' position, reducing incorporation efficiency. To address this, researchers have investigated engineering TdT variants with expanded substrate pockets, although no commercially optimized mutant has yet emerged.
Figure 2. (A) Two-step extension synthesis of DNA oligo using TdT and dNTPs with reversible blocking groups. (B) Two-step extension synthesis of DNA oligo using TdT–dNTP conjugates for reversible termination of oligo elongation[2].
An alternative orthogonal protection strategy was developed by Palluk et al. (2018) that covalently couples TdT to a dNTP. Once this conjugated nucleotide is incorporated into the growing chain, the attached TdT effectively blocks further extension. Cleaving the TdT from the primer thus resets the chain for the next cycle. Not only does this approach bring coupling times down to 10–20 seconds, but it also affords a highly-controlled process that is a highly attractive direction for scalable EOS platforms.
An alternative EOS strategy employs template-dependent polymerases such as high-fidelity DNA polymerases or engineered reverse transcriptases. These enzymes can extend DNA primers only in the presence of a short, transiently hybridized template strand. Hoff et al. (2019) demonstrated a technique using two-nucleotide templates that transiently bind to the primer, guiding single-base extension through reversible terminators.
The extension cycle mimics that of reversible terminator sequencing: (1) incorporation of a 3'-blocked dNTP guided by transient hybridization, and (2) deprotection to expose the 3'-OH for the next round. This approach achieves impressive base-calling accuracy (>98% stepwise efficiency) and was used to synthesize 20-mers within minutes. Importantly, unlike template-independent systems, this method allows sequence-specific synthesis using standard enzymes and natural nucleotides, eliminating the need for extensive enzyme engineering.
Figure 3. Template-dependent polymerase mediated oligo synthesis by transient hybridization and chemically blocked substrates[2].
EOS has wide-ranging and potentially game-changing applications. It allows for the creation of longer, purer and more structurally varied DNA constructs that cannot be made by chemical synthesis or that are prohibitively difficult or expensive to make chemically. Two particularly exciting applications of EOS are whole-genome synthesis and DNA data storage.
Whole-Genome de novo Synthesis
EOS can provide the scalability and fidelity needed to make de novo genomes. For years, chemically synthesized genomes have enabled the move from viral genomes all the way to complete synthetic eukaryotic chromosomes. With EOS, short oligos can be assembled into gene-length fragments and full genomes using PCR and DNA assembly methods. In recently published work from Venter Institute and others, bacterial genomes have been synthesized and 'booted up' in host cells for use in studying minimal life and the function of entire genomes. EOS makes the environmental impact and reagent cost of genome synthesis drop by multiple orders of magnitude. This makes it scalable to large genomes and applications.
DNA Data Storage
As digital information volume explodes, DNA has emerged as an ultra-dense, stable, and sustainable medium for long-term data storage. EOS enables writing arbitrary sequences with high accuracy, providing a path to encode digital information into DNA at industrial scales. The compatibility of enzymatic systems with miniaturization and automation further enhances their potential for use in DNA-based data centers.
Figure 4. The principle of DNA data storage. Three key technologies are required: (a) encoding and decoding technology, which encodes binary strings into DNA strings and decodes DNA strings back into binary strings. (b) DNA synthesis technology for the actual data writing process. (c) DNA sequencing technology for the data reading process[2].
The standard workflow for de novo genome synthesis includes:
EOS is most beneficial in Step 2A, where it can reduce costs, increase yields, and improve sequence quality.
For affordable, high-throughput DNA synthesis in the future, all of these EOS-related steps, from nucleotide synthesis, base incorporation, cleavage, sequence verification, and error correction need to be fully automated on a single platform. Integrating EOS and CRISPR-based base editing would also allow for on-the-fly correction of synthesis errors during the DNA synthesis process itself.
Alfa Chemistry will continue to work on enzymatic synthesis by providing new enzymes, reagents, and integrated platforms for DNA manufacturing, helping the biotechnology community advance the field of synthetic biology and genomic engineering.
Alfa Chemistry provides a diverse library of oligonucleotide synthesis building blocks to enable researchers to design and synthesize their own custom oligonucleotides. The key intermediate building blocks with their tightly controlled purities and physicochemical properties, for seamless implementation in automated SPOS workflows. The use of these pre-functionalized synthetic units allows for a dramatic reduction in synthetic complexity and synthesis yields and also allows for modular design of complex oligonucleotides.
1. Can enzymatic oligonucleotide synthesis be used to synthesize RNA sequences?
Yes, with engineered RNA polymerases or reverse transcriptases, EOS principles can be adapted to RNA synthesis, though it remains more challenging due to ribose 2'-OH sensitivity.
2. What are the current length limits for EOS-synthesized oligonucleotides?
Typically, EOS enables synthesis of 20–60 nt oligos with high fidelity. Longer sequences require fragment assembly post-synthesis.
3. How does EOS compare in cost to phosphoramidite synthesis?
EOS has the potential for lower cost per base due to aqueous conditions and reduced waste, but currently requires specialized reagents and enzymes that may offset savings.
4. Are enzymatic oligonucleotides suitable for diagnostic or therapeutic use?
Yes, their high purity and reduced damage profile make EOS products ideal for sensitive applications like qPCR, CRISPR, and antisense therapeutics.
5. What is the environmental advantage of enzymatic synthesis?
EOS uses water-based chemistry, eliminating toxic solvents and significantly reducing hazardous waste compared to traditional methods.
6. Can EOS support modified bases or unnatural nucleotides?
Some EOS systems can incorporate base analogs with engineered polymerases, but compatibility depends on the enzyme and blocking strategy used.
References
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