Enzymatic DNA synthesis may be a still some way off, however the route to overcoming the DNA synthesis bottleneck is clearer than ever.
In emerging fields like personalized medicine and synthetic biology, DNA is the major stock in which companies trade. Molecular techniques to engineer DNA have moved in lockstep with the ability to sequence DNA. These techniques are adapting to automated platforms, allowing for highly accurate, scalable functional assays and production methods. Now, a major bottleneck reveals itself – DNA synthesis.
Molecular biology: Past to present
Forty years ago, Sanger sequencing and Polymerase Chain Reaction (PCR) opened molecular biology up to a new world of applications in personalized medicine, forensic science and genetic engineering. In the 1990s, miniaturization and better technologies gave us higher throughput versions of these techniques which saw the completion of the Human Genome Project completed ahead of schedule. New methods evolved, like real-time PCR and Next Generation Sequencing, with rapid turnaround times – hours rather than days – revolutionizing diagnostics. DNA synthesis, however, has remained stuck.
The phosphoramidite method of DNA synthesis was first developed in the early 1980s and is still used today. The process assembles single strands of DNA using synthetic building block versions of the A-T-G-C bases of DNA. A single strand of DNA is assembled in a stepwise manner, base by base, with chemical “de-capping” steps after each base is added to allow the next to bind. The process is arduous, not least because the error rate is 1 in 200. These errors cannot be detected during synthesis, meaning several short strands must be constructed in parallel to ensure a correct sequence is made.
Biotechnology is getting smarter. Automated platforms and engineering approaches in the emergent field of synthetic biology is reshaping R&D and breaking new ground for molecular biology in gene therapy and data storage. With all the advanced molecular tools at our disposal, DNA synthesis remains the last great stumbling block. Leaders of the next biotechnology revolution, including Microsoft and Ginkgo Bioworks, are making deals with DNA suppliers measured in base pairs.
Scale down to scale up
Waiting 4-8 weeks for a novel gene, bespoke sequence or DNA pool to be synthesized no longer cuts the mustard when DNA cloning occurs in a day and the sequence can be determined overnight. Recognizing the need to de-bottleneck molecular biology, several companies are developing exciting new technologies.
Leading the field is Twist Bioscience. Twist have developed a miniaturized version of the phosphoramidite cycle on a silicon wafer, like a computer chip. This chip anchors tens of thousands of strands of DNA for synthesis, which can be assembled in situ. This vastly increases the capacity compared to conventional approaches. Twist has seen major success with this method, with strategic partnerships with companies like Ginkgo Bioworks to provide over one billion nucleotides of DNA – that counts for 10% of the global DNA synthesis market. Twist have almost doubled their stock price since their IPO in October 2018.
Evonetix are taking a different approach to scaling DNA synthesis. They have developed a silicon chip with a microelectromechanical system, like the circuitry on a computer chip, giving them control over individual spots on the surface. Using heat to create a “virtual well” in liquid on the chip’s surface, they can regulate the phosphoramidite cycle at precise spots using thermal control rather than the current chemical-based cycle. Because the technology is miniaturized, they are developing their technology as a DNA writer for the laboratory bench.
Evonetix have also developed a quality control system whereby two complementary strands of DNA are allowed to bind. If there is a mismatch in either strand, the melting point of the DNA molecule is lower than a perfect match. Using a simple temperature check, they can test their constructs on-chip for errors. This tight thermal control may have the added bonus of being future-proof as the next big approach to DNA synthesis requires precise temperatures to work – enzymatic DNA synthesis.
Life finds a way
For decades, researchers have used naturally occurring enzymes to replicate DNA. The DNA polymerase enzyme of Thermus aquaticus, a bacterium found in hot springs, gave us Taq Polymerase which survives the high temperatures of DNA denaturation at the beginning of each PCR cycle. More enzymes have been found over the years, but all have the same function – to bind to a DNA template and copy a strand. Some enzymes even have proofreading function to ensure they have placed the correct nucleotide pair together. Standard Taq polymerase can go thousands of bases without making an error, already an improvement on the phosphoramidite cycle, but then Taq cannot make DNA from scratch.
To get around the template issue, nature has once again provided us with a solution. Terminal deoxynucleotidyl transferase (TdT) is essentially a DNA polymerase that works without a template. In humans, TdT plays a role in antibody gene recombination where it adds non-templated nucleotides to non-coding regions in antibody genes, allowing for our unique and diverse immune systems. In 2018, Harvard geneticist George Church and his team successfully encoded and decoded brief messages using enzymatically synthesized DNA.
In the space of a year, three companies started developing this nascent technology. California based Molecular Assemblies were one of the first in the field. Their enzymatic DNA synthesis approach promises to decrease post-synthesis processes required by current technology, as well as increase length of DNA strands by 10-50 times that of the current chemical method. The enzymatic approach is also far more sustainable, as the phosphoramidite method uses several toxic compounds in de-capping bases.
French company DNA Script also show much promise, synthesizing a 150 base length last year with their approach. They are quick to highlight the lower error rate and faster delivery time compared to conventional DNA synthesis companies. Earlier this year, DNA Script raised $38.5 M in Series B funding. Another two notable players in the field are Ansa Biotechnologies and Nuclera, highlighting the enthusiasm for this technology.
Enzymatic DNA synthesis may be a still some way off, however the route to overcoming the DNA synthesis bottleneck is clearer than ever. Like DNA sequencing before it, miniaturization and scaling reflected in the medium-term solution, while game-changing technologies are developed for the long-term, just in time for the industrial biotech revolution.
