Team Talk – Dr Stuart Crosby, Head of Synthetic Chemistry

Dr Stuart Crosby has over 20 years’ research experience working in the fields of synthetic chemistry and drug discovery. He is now Head of Synthetic Chemistry at Evonetix where he is developing the chemistry that underpins Evonetix’s third generation DNA synthesis platform.

I joined the Company in 2015, right at the beginning when it was spinning out from Cambridge Consultants. My background is as a pharmaceutical chemist, but I was looking for a new career direction, so it was hugely exciting to be one of the first chemists on the team and to develop a new research program.

As the first chemists in the building, we had the opportunity to set up the lab from scratch, which was an interesting challenge! Initially, it was the technology itself that attracted me to the Company, I could see the potential it had to revolutionise synthetic biology, and therefore the world, and I wanted to contribute my expertise as a chemist and be a part of building it, from initial proof of principle through to commercial reality.

The focus of my role has been on developing the chemical processes that underpin the oligonucleotide synthesis and DNA assembly on our platform, to enable the building of double stranded DNA products of much greater length, quality and diversity than anything that has been achieved before.

My role has changed over the years. Our initial work focussed on proving that the fundamental chemistry could work. As the company has grown and evolved the team has become completely interdisciplinary, and while my focus is still on the chemical processes, this now involves working with different groups across the Company, including biologists, engineers and physicists. This progression has also seen my work as a chemist change, transitioning from chemistry in test-tubes and flasks to the surface of our own Evonetix silicon chip.

When I first joined Evonetix, synthetic biology was still developing as a concept, albeit one with massive potential, so the Company’s technology was attractive because it was completely novel and cutting-edge. I was interested in the idea of applying engineering principles to biological systems and the potential impact of synthetic biology applications were extremely exciting.

One of the biggest challenges was in having a such a novel DNA synthesis platform to carry out the oligo synthesis and DNA assembly on. The surface of our silicon chip is covered with many thousands of reaction sites where chemical reactions can be controlled by changes in temperature. To allow controlled oligo synthesis in these “virtual wells” we have had to develop reactions which are highly temperature sensitive. However, the commercial phosphoradamite process used for growing oligos on columns has been pretty much unchanged for the past 40 years, so upgrading this process to be compatible with our platform, was a huge undertaking.

Another major challenge was in understanding how reactions could even be designed to be highly thermosensitive. Since no-one has ever had a platform like ours, where a reaction must proceed very, very slowly at cold temperatures and very quickly at hot temperatures, the literature precedent in this area was sparse and even contradictory. A Rule-of-Thumb that every chemist knows from their undergraduate days states that reactions in solution double their reaction rate per ten degrees of increased temperature. We have been able to demonstrate that this rule is completely wrong – which is lucky otherwise our technology would not work!

Again, collaboration was key; especially for understanding the fundamental reaction kinetics needed for the thermal control to work. We worked closely with physicists and engineers who developed the scripts and equations for us to apply to the chemistry. We could then input raw experimental data from reactions carried out at different temperatures into these scripts to gain insight into the reaction kinetics. This helped us understand crucial aspects of our synthetic process such as the error-rate at hot and cold sites and optimum reaction time for thermally controlled steps.

Optimising the duration of each step is critical to the performance of Evonetix’s synthesis approach.

We are growing DNA on a miniscule scale – our reaction sites are of micrometer diameter and are only covered in a single layer of oligos – just femtomoles of DNA! That’s fine in our final product since by its very nature DNA is replicable, and we can amplify and sequence the resulting DNA products. In early development however, we needed to circumvent the fact that we weren’t make enough oligo to directly analyze it using conventional chemistry lab instrumentation. We got round this problem in a couple of ways: One method was to use fluorescently tagged complimentary oligos which hybridise with correctly synthesised sequences on reaction sites. These sites then light up when viewed with a fluorescent microscope.

Another way of testing our chemistry on a scale at which standard analysis, such as LC-MS, could be used was to make our own column-based oligo synthesizers. These were much like the commercial models currently used throughout the industry but with one crucial difference – the column could be either heated or cooled accurately. This effectively replicated conditions at a single reaction site but on a massively increased scale so that the oligos and any side-products could be easily identified and the chemistry could be optimised.

A large part of tackling this challenge was to work closely with the other disciplines to investigate the logistics of automating our chemistry process– what microfluidic valves we should use, the best flow rates and so on. It’s also important to think about how we analyse the chemistry, which involves working with the physicists and biologists to determine how to cleave the DNA products from the reaction sites for analysis. It has required some very novel thinking on all aspects of the platform design.

One of the most fundamental challenges is the one we are trying to address – access to DNA. Currently DNA is relatively easy and inexpensive to read and edit thanks to Next-Generation-Sequencing (NGS) and CRISPR. But it is still very costly and labour-intensive to write long, gene-length DNA at the low error-rates required for incorporation into biological systems. There has not yet been the orders-of-magnitude drop in the cost and time for DNA writing that NGS delivered for DNA reading, and the only way to do that is to make DNA in a massively parallel way with an automated error-detection/removal process.

I believe that biologics will increasingly take over from small molecules in pharmaceutical chemistry, and this will be further accelerated by synthetic biology – A great recent example of this is the development of the mRNA vaccines for SARS-CoV-2. However, the future possibilities reach into many fields, for example synthesis of fuels and materials, biosensing, artificial cells, decarbonisation and improved food supply.

Our platform has an enormously broad range of potential applications. The technology is addressing a fundamental cornerstone that underpins the whole field of synthetic biology, so we could be supporting research into pharmaceuticals, precision medicine, fuels, materials, all the way through to data storage and food security. We will likely see the impact in pharmaceuticals first, as there are already toolkits ready to apply this technology. But further than that, we will be sure to witness exciting developments across many disciplines.

For more information about Evonetix, please visit: www.evonetix.com.