Enzymatic Approaches Will Transform DNA Synthesis, but Which One Is Right for You?

The following article is an opinion piece written by Dan Lin-Arlow. The views and opinions expressed in this article are those of the author and do not necessarily reflect the official position of Technology Networks.

After decades of incremental improvements, the DNA synthesis space is finally on the verge of a significant leap forward. It’s high time – scientists have been stuck with the same old phosphoramidite chemistry and its inherent limitations in length, speed, accuracy and environmental impact for too long.

There has been much discussion lately about the promise of a newer approach known as enzymatic DNA synthesis, which has been billed as the solution to problems associated with traditional chemical synthesis.

However, with several scientists and companies developing different enzymatic methods, it can be a challenge to understand the differences between new offerings. As vendors vie for attention, it’s important to know which factors to consider before choosing the one that best meets your scientific needs.

First, it’s worth taking a moment to lay out the flaws associated with traditional DNA synthesis methods. Chemical synthesis tends to be relatively slow, taking several minutes to add a single base. Second, because the reagents used in the process gradually damage the growing strands of DNA, the cycle can only be performed a certain number of times before the majority of molecules contain lesions. Therefore, chemical synthesis can only produce relatively short oligos.

For gene synthesis, i.e., construction of double-stranded DNA molecules longer than a few hundred base pairs, several oligos must be assembled, but the assembly process can potentially introduce errors into the final sequence. Additionally, the toxic material needed for synthesis creates hazardous waste that must be disposed of, leading to additional costs and the risk of environmental harm.

Despite these problems, perhaps the most significant issue with chemical synthesis today is that it is unlikely to improve much further. Scientists have been refining these processes for decades, and where we stand now seems to be at the limit of what’s possible with phosphoramidite chemistry.

In contrast, enzymatic DNA synthesis harnesses the power of biological enzymes to do what they do best: synthesize DNA efficiently and accurately without needing or creating any hazardous materials. Scientists have explored various enzymes and engineering approaches as the foundation for enzymatic synthesis, and there is plenty of room for continued improvement.

If you’re interested in considering an enzymatic alternative to traditional DNA synthesis, there are several factors to consider.

Speed

Because new approaches are based on differently engineered enzymes, the time to add a single base varies substantially between vendors. This influences how quickly the product can be delivered to the end user.

Oligo length

While some enzymatic approaches produce oligos as short as (or even shorter than) their chemical synthesis counterparts, others are setting new records in the length of oligos synthesized. Of particular importance in gene synthesis is the use of longer oligos, which means that fewer parts are required to construct a sequence of a given length, enabling greater reliability and flexibility in the types of sequences that can be assembled.

Complexity

Gene synthesis vendors that assemble long sequences from chemically synthesized oligos have conditioned scientists to accept that many sequences are too “complex” to be manufactured because they contain regions of high or low GC content, repeats or hairpins.

However, some enzymatic methods that can directly produce long oligos greatly facilitate the manufacturing of complex sequences, so scientists no longer have to compromise the designs of their experiments. If you’ve had gene synthesis orders rejected for being too complex in the past, look for a vendor with an enzymatic technique that enables the production of complex DNA.

Mass

Yield differences across platforms can lead to higher – or lower – mass of oligos produced. While this may not matter for gene synthesis projects, it's important to keep in mind if your application requires large amounts of material, such as for an oligonucleotide therapeutic.

Currently, chemical synthesis has the cost advantage due to an established supply chain with scaled-up manufacturing of building blocks, although this could change in the future.

Modifications

Over phosphoramidite chemistry’s long history, an extensive catalog of modified DNA building blocks has become available, including monomers with modified bases, sugars, fluorophores/quenchers, linkers and tags. Existing enzymatic synthesis providers typically only make unmodified oligos or offer a limited catalog of modified bases. Chemical synthesis still has the advantage for applications requiring highly modified oligos.

Instrument vs. service

For the first time, scientists now have the option of a benchtop instrument that can perform enzymatic synthesis of short oligonucleotides in their labs. This may be convenient for scientists interested in a certain range of applications. However, in some cases, the turnaround times achieved by the leading synthesis services rival those of the benchtop instruments for equivalent products.

Furthermore, unlike the instruments, the services provide rich QC data about the oligos produced. The oligos produced by these benchtop instruments are also not ideal for longer and more complex gene synthesis because they are relatively short. Finally, biosecurity is a concern; it’s easier to monitor and control which sequences are made at a factory than to secure the synthesizers in all customers’ labs.

We’re in the early days of enzymatic synthesis, and chemical synthesis can still result in a better product for some applications. However, with so much room for improvement, it seems clear that the precise and environmentally friendly enzymatic approach will ultimately outperform traditional synthesis and allow for the production of a broader range of DNA sequences than has been possible with legacy technology.