Artificially synthesized sequences of DNA and RNA are an integral part of the toolbox used in biopharmaceutical innovation. They have long been used in molecular biology for a wide range of applications, including PCR, cloning, DNA sequencing, and a variety of assays that detect gene expression. Now, DNA synthesis is in high demand in newer applications like CRISPR. In certain cases, such as antisense and RNA Interference, the oligonucleotide can itself be a drug. To meet these needs, manufacturers must make synthetic oligonucleotides (oligos) in scales ranging from microgram amounts of tens of thousands of different sequences in parallel to single batch kilogram lots of drug product.  

Two approaches exist to make synthetic oligonucleotides: chemical and enzymatic synthesis. For many years, chemical synthesis has been the dominant manufacturing technology. The reagents and synthesis protocols have been highly optimized since its introduction over 40 years ago. It is used today to manufacture oligonucleotides with a wide variety of chemical modifications in yields ranging from sub-nanogram to ten-kilogram scales. Enzymatic synthesis, by contrast, is only now entering the market in a significant way, and the technology is rapidly evolving. It is likely that both chemical and enzymatic methods will play major roles in future oligonucleotide manufacturing. 

Chemical synthesis offers a few key advantages that have made it the dominant choice for many applications. It is done by building the DNA sequence one base at a time from the 3’-end stepwise with phosphoramidite monomers. Phosphoramidites are reactive analogs of nucleotide monophosphates in which functional groups that do not participate in the elongation of the oligonucleotide are protected with labile protecting groups. The protecting groups are needed to tightly control the reaction between the phosphoramidite monomers and the growing DNA oligo in order to maintain high sequence specificity.

The addition of each monomer involves four sequential chemical steps: deprotection, coupling, capping, and oxidation. This process has built-in measures that help prevent the synthesis of undesired molecules and allow the correct DNA chain to continue to grow until synthesis is complete. At that time, the sequence is cleaved from the solid support, and the protecting groups are removed, leaving the final desired oligonucleotide sequence. Over the past four decades, many innovations and process optimizations have been achieved, which have led to the current state where the coupling efficiency of each base addition proceeds at >99% efficiency, an amazing level of efficiency for bulk organic chemistry reactions. Following each cycle, a tiny amount of unelongated product is present due to failure to react with the phosphoramidite monomer. These unelongated failure products are terminated at the capping step, which prevents the continuation of synthesis after a coupling failure and thus helps prevent internal deletions and makes purification easier after synthesis since most of the failure products are relatively short and are easily separated from the full-length desired product. If capping were not part of the cycle, there would be more failed products that are missing a single base, which is more difficult to purify away from the full-length product. For long oligos, the unwanted failure products can overshadow the desired full-length product. For example, a 200-mer oligonucleotide requires 199 coupling steps. At 99.0% efficiency, that translates to 0.99¹⁹⁹, which means only 13.5% of the material synthesized is full length. Even at 99.6% efficiency, only 45% of the final product is full length. Can enzymatic synthesis do better than this?

‍Pros and Cons of Enzymatic Synthesis

Enzymatic synthesis differs from chemical synthesis in several ways, the first being that it more closely reflects a natural process in living cells, where most DNA synthesis is driven by DNA polymerase enzymes. However, that process requires a template nucleic acid sequence as a blueprint to direct the order of base additions. In enzymatic oligonucleotide synthesis, different enzymes must be used that instead enable the sequential stepwise addition of bases in any desired order without a template, building from the 5’-end. Most enzymatic synthesis work to date has focused on the use of terminal transferase (TdT) as the tool to build DNA, although other enzymes are now beginning to emerge (for example, poly-U polymerase or PUP).

TdT is a naturally occurring enzyme that functions in T cells and B cells to add extra bases to DNA sequences ⁽¹⁾ during the recombination events that create functional antibodies or T-cell receptors. Enzymatic synthesis makes use of the natural ability of this enzyme, adding nucleotides to DNA one base at a time to manufacture synthetic single-stranded DNA. Significant effort has gone into the development of reagents and methods that permit single base addition using TdT—without modification, the enzyme would incorporate a random sequence of bases into the nascent oligonucleotide of variable length. As part of this endeavor, mutant TdT enzymes have been evolved that can efficiently utilize the modified substrates needed to enforce single-base stepwise addition. TdT can also be mutated to enable the use of other chemical modifications, such as modified RNA residues (e.g., 2’-OMe RNA, 2’-F RNA, etc.) or non‑base modifiers like fluorescent dyes or biotin. The natural wild-type TdT enzyme often works poorly or not at all with highly modified substrates. 

One of the advantages built over the decades of use of chemical synthesis is a huge repertoire of chemical modifications that work with the phosphoramidite synthesis method. Only a limited number of modifications are currently available for use in enzymatic synthesis methods. It will likewise take years of development work to attain a similar level of functionality using enzymatic synthesis. One possibility is to develop in parallel a suite of different enzymes that can function on subsets of modified bases or non-base groups. This will require a delicate balance between altering the enzyme to accept a modified substrate while retaining its ability to work efficiently and with high specificity.

The single most significant advantage of enzymatic oligo synthesis is that it permits manufacturing in an aqueous environment without using toxic organic chemicals that comprise a significant hazardous waste burden. Large-volume chemical synthesis is usually done at centralized plants that are built to handle hazardous waste. Without producing large volumes of organic waste, enzymatic synthesis opens the possibility of performing DNA synthesis anywhere, on a lab benchtop ⁽²⁾ or even in the field.

Another potential advantage of enzymatic synthesis is that it may offer the possibility to make even longer DNA sequences than are made today. The utility of chemical synthesis starts to decline around 200-mers, making it challenging to use the process for directly synthesizing genes or gene fragments. ⁽¹⁾ The longest direct-synthesis oligonucleotides in routine production today are around 400-mer in length. While longer oligos can be made, the small amount of desired full-length product present makes these of limited utility. Today, longer products (genes or gene fragments) are typically made using assembly methods that start with short to long chemically synthesized oligos and combine them using ligase or polymerase enzymes. 

A number of groups are working to improve the coupling efficiency of enzymatic synthesis and to make the process commercially viable. A list of several companies working in the enzymatic oligonucleotide synthesis space is shown in Table 1. Some groups have reported a coupling efficiency as high as 99.7% to 99.9%, which exceeds the best coupling efficiency of the phosphoramidite chemical method and may enable direct synthesis of 1000-mers or longer.

Some additional issues unique to enzymatic synthesis remain to be solved. For example, one risk of TdT‑based synthesis performed in aqueous conditions is that as the sequences get longer, DNA secondary structures can form that interfere with enzyme access and can decrease coupling yields. These structures cannot form in a chemical synthesis environment, where the bases are in an organic buffer and are modified with protecting groups. A research group led by the Joint BioEnergy Institute in California reported that they were able to limit the impact of certain secondary structures during enzymatic synthesis by reducing the divalent ion concentration while simultaneously increasing the reaction temperature. ⁽¹⁾

Another issue that remains to be solved involves product purity or, rather, the nature of the impurities produced during synthesis. Enzymatic synthesis does not incorporate a capping step like chemical synthesis does. After the coupling step in chemical synthesis, molecules that fail to extend are capped with an acetylating agent to prevent continuing synthesis on defective molecules. The capped failure products are short and easy to remove from the final product. ⁽³⁾ In enzymatic synthesis, by contrast, the lack of a capping step allows failure products to continue to grow. Hence, the dominant failure products are largely heterogeneous “n-1-mer” species, which are comprised of oligos that are missing a single base at every position. These failure species are long and thus are more difficult to separate from the desired end‑product, which is only a single base longer.

Reagent and production costs are yet another aspect of enzymatic synthesis that must be improved before this approach can gain widespread use. Chemical synthesis is almost always performed using phosphoramidite building blocks and the same reagents are used by all major commercial manufacturers. As a result, these critical reagents are made in large bulk lots, allowing for significant cost savings through economies of scale. Most enzymatic oligonucleotide synthesis companies utilize their own proprietary substrate building blocks, and hence, no universal reagent set exists that can be made on a similar scale. Another less obvious cost barrier relates to synthesis cycle time. Modern high-throughput chemical synthesis is typically done in a high-efficiency commercial environment that depends on rapid cycle times to enable many oligonucleotides to be manufactured on a single instrument every day. If an average base addition cycle time for phosphoramidite chemistry is 3 minutes but for enzymatic synthesis, it is 9 minutes, then a facility will need 3x the number of synthesis machines and 3x the physical space/footprint to meet demand if converted to enzymatic synthesis. Hence, COGS is not simply the raw reagent cost—it also involves many other factors. Cost savings will be realized with enzymatic synthesis by reducing the need for safe organic waste disposal.

‍The Future of Enzymatic Synthesis

Once the issues with enzymatic synthesis are worked out, it could become the go-to method for certain applications. CRISPR gene editing, for one, requires long DNA templates to provide instructions to make precise and often large changes to the genome. The process of developing and manufacturing antisense drugs could also benefit from the ability to make oligos with enzymatic synthesis, enabling the manufacturing of multi-kilogram quantities of these short compounds without producing large volumes of organic chemical waste.

Enzymatic synthesis is still in its infancy, but it is rapidly evolving and offers the potential to expand both the access to DNA synthesis and the potential applications for it. Given the different advantages and disadvantages of chemical and enzymatic approaches, biopharma developers would be well-served by finding ways to exploit their complementary nature. For example, they could use chemical synthesis to make highly modified oligos on a large scale while at the same time using enzymatic synthesis to make small-scale, unmodified oligos or large-scale oligos with a more limited suite of modifications. ⁽⁵⁾

To make that vision a reality, the industry will need to rethink its design of DNA synthesis facilities. The ideal plant of the future might offer a hybrid format, employing both chemical and enzymatic methods. It will not be easy, but it will be essential to ensure that genomics researchers can find the right path forward for any innovation they dream up that requires efficient, affordable, and precise DNA synthesis.

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References

1. Source: Eisenstein, M. Enzymatic DNA synthesis enters new phase. Nat Biotechnol 38, 1113–1115 (2020). https://doi.org/10.1038/s41587-020-0695-9.
2. Source: Barthel S, Palluk S, Hillson NJ, Keasling JD, Arlow DH. Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis. Genes. 2020; 11(1):102. https://doi.org/10.3390/genes11010102.
3. Source: Jensen, M, Davis, R. Template-Independent Enzymatic Oligonucleotide Synthesis (TiEOS): Its History, Prospects, and Challenges. Biochemistry 2018, 57, 12, 1821–1832. https://doi.org/10.1021/acs.biochem.7b00937.
4. Source: Lennox, K.A., Behlke, M.A. A Direct Comparison of Anti-microRNA Oligonucleotide Potency. Pharm Res 2010, 27, 1788–1799. https://doi.org/10.1007/s11095-010-0156-0.
5. Source: Ashley, J, Potts, I, Olorunniji, F. Applications of Terminal Deoxynucleotidyl Transferase Enzyme in Biotechnology. ChemBioChem 2023, 24. https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202200510.

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About the author

Mark Behlke, MD, PhD, is the Chief Scientific Officer at Integrated DNA Technologies.