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Reshaping Biology with Enzymatic DNA Synthesis

    The blueprint of life is written in DNA

    Cells can perform incredible feats. On the smallest scale, they rearrange atoms and generate nanometer-sized machines like motors and turbines [1]. On a large scale, they shape the surface and atmosphere of our planet. And it’s thanks to the staggering cooperation of about 30 trillion cells jointly forming a human body [2] that you can decipher these very words.

    The basics of all these functions are, of course, programmed in DNA — the well-recognized, double helix-shaped polymer which consists of four different nucleotide subunits. DNA was discovered in 1869 by Friedrich Miescher, but it wasn’t until almost a century later in 1953 that its structure was resolved by James Watson, Francis Crick, Rosalind Franklin, and Maurice Wilkins [3].

    The early days of DNA synthesis

    At around the same time, the first man-made DNA molecule, a thymidine dinucleotide, was created in the laboratory [4], laying the foundation for the field of DNA synthesis. Over the following decades, a small community of chemists developed various iterations of reaction schemes to increase the lengths of synthetic DNA molecules that could be synthesized, ultimately establishing the nucleoside phosphoramidite method in 1981 [5]. To this day, phosphoramidite-based synthesis remains the primary method for DNA manufacture and is often referred to as “chemical DNA synthesis”.

    Interestingly, despite the essential role DNA plays in biology, it took the scientific community a long time to appreciate the importance of synthetic DNA. Marvin Caruthers, the inventor of chemical DNA synthesis, reported that “there were no biologists, biochemists or molecular biologists anxiously waiting for [them] to develop [the technology]” [6]. This perception didn’t even change when H. Gobind Khorana synthesized the first gene and was awarded the Nobel Prize in Physiology or Medicine in 1968 for the interpretation of the genetic code and its function in protein synthesis [7]. Nature New Biology commented that “Like NASA with its Apollo Programme, Khorana’s group has shown [that synthesizing a gene] can be done, and both feats may well never be repeated…”. [6]

    Synthetic DNA unlocked life sciences research as we know it

    Fast forward to the present, just a few decades later, and we are engineering metabolic pathways in cells to turn them into factories for valuable chemicals, are developing personalized cell-based therapies to treat cancer and have just overcome a global pandemic using an mRNA vaccine — all powered by our ability to write DNA sequences. No human has set foot on the moon since the Apollo Program finished in 1972, but scientists use millions of genes every year.

    This trajectory of the field of biology would have been nearly impossible to predict. Keystone technologies empowered by synthetic DNA oligos, such as PCR amplification [8] and DNA sequencing [9], have enabled scientists all over the world to copy and read DNA. More recently, CRISPR-Cas9-based genome editing guided by synthetic RNA oligos has led to an incredible increase in the rate of scientific progress [10].  

    Caruthers and his colleagues therefore showed remarkable foresight. They understood earlier than everyone that the ability to write DNA — the code of life — would be immensely powerful. And that handing such technology to the scientific community would enable accomplishments beyond anyone’s imagination.

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    Pushing the boundaries of DNA synthesis further

    I believe a similar logic applies today to the improvement of DNA synthesis as an omnipresent biological tool, prompting our pursuit for the next generation technology.

    While phosphoramidite chemistry has been a workhorse for many applications, it’s insufficient for the life sciences industry of the future. To truly unlock the genomics revolution, employ synthetic biology for sustainable food and chemical production and make groundbreaking advances in healthcare, we need to drastically improve our ability to write DNA.

    Reliable access to long DNA constructs of any sequence — think 10 kb, within 5 days — could double to quadruple the rate of research progress of gene and cell therapy companies, according to a survey we performed. At a recent microbiome conference, over 100 scientists attended our round table discussion on “complex” DNA sequences which are difficult to make using phosphoramidite synthesis, two-thirds of whom had encountered issues obtaining the DNA constructs they need. 

    Building complex biological systems requires rapid iterations, and accelerating this cycle of learning and improvement is essential for realizing the promise of engineering biology. Can you envision how life sciences research would change if any DNA construct could be synthesized in one day? Or, imagine what you could do with custom-synthesized libraries of millions of genes or even plasmids for screening applications. A world where the artificial term of a complex sequence has disappeared, and any biologically important DNA construct can be generated rapidly. 

    To unlock this, a real upgrade for writing DNA sequences is required. Incremental improvements to the current chemical process, applying new techniques for the assembly of short oligos, and band-aid solutions for the isolation of correct molecules won’t suffice. We need a paradigm shift — a method for writing DNA sequences that outperforms the current state of the art by a factor of 5, 10, and ultimately 100.

    The future of DNA synthesis will be enzymatic

    Here is where enzymes enter the equation. 

    Enzymes catalyze chemical reactions at unmatched efficiency by selectively binding molecules and arranging them with picometer precision to initiate the formation of new chemical bonds. If you want to see this magic happen within the context of de novo DNA synthesis, check out PDB structure 4I27, which shows a TdT enzyme binding a single stranded piece of DNA and an incoming nucleotide (dNTP). Enzymatic catalysis allows for perfect specificity, the ultimate requirement to enable the next generation of DNA synthesis.

    In contrast, chemical synthesis requires 10,000-fold higher concentrations of reagents and operates by forcing molecules to randomly collide with each other. Activating and protecting groups are used to guide the process towards the formation of desired bonds, but unavoidable side-reactions occur that damage the growing strands, limiting the quality of product that can be achieved. This sets a ceiling for chemical DNA synthesis which is orders of magnitude below what enzymatic approaches can accomplish.

    Using enzymes is only part of the trick, though. Dozens of attempts at developing enzymatic DNA synthesis technologies haven’t been able to clear the high bar set by phosphoramidite synthesis. It really comes down to how those enzymes are put to work [11]. It requires building a world-class interdisciplinary team of scientists and engineers, capable of solving a multitude of technical challenges. It comes down to securing sufficient investment to support the respective development effort.

    Accelerating life sciences

    About 5 years into our journey at Ansa Biotechnologies, we can proudly say that we have succeeded at building an enzymatic DNA synthesis technique superior to the phosphoramidite method. We have pushed synthesis lengths far beyond previous limitations, unlocking feats such as the direct synthesis of entire, short plasmids as single oligonucleotides. We can perform synthesis at scale and have just written the complete sequence of Yeast chromosome 1 in 230 oligos spanning about 1kb each, on a fraction of one instrument run, in under 2 days.

    This is just a first glimpse at the next generation of DNA synthesis and what it will enable. Our initial focus is on making access to DNA constructs, including complex sequences, much easier and faster. We want to free scientists from spending their time on tedious molecular cloning work and remove the restrictions legacy DNA vendors impose on R&D progress. But we won’t stop there — our platform technology could benefit the scientific community in many application areas that we will discuss here in the future, including the generation of novel types of libraries, and manufacturing of nucleic acids beyond DNA.

    Our mission at Ansa is to accelerate life sciences progress, and to support you in making the next scientific breakthroughs.

    By Sebastian Palluk, Ansa CTO and co-founder

    1. Sambongi Y, Iko Y, et al. 1999. Mechanical Rotation of the c Subunit Oligomer in ATP Synthase (F0F1): Direct Observation. Science. Vol 286, Issue 5445
    4. Michelson AM, Todd AR. 1955. Nucleotides part XXXII. Synthesis of a dithymidine dinucleotide containing a 3: 5-internucleotidic linkage. Journal of the Chemical Society. 0(0):2632–2638
    5. Beaucage S, Caruthers M. 1981. Deoxynucleoside phosphoramidites – A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters. 22(20):1859–1862
    6. Caruthers MH. 2011. A brief review of DNA and RNA chemical synthesis. Biochemical Society Transactions. 39(2):575–580
    7. Khorana HG. 1979. Total Synthesis of a Gene. Science. Vol 203, Issue 4381
    11. Palluk S, Arlow DH, et al. 2018. De novo DNA synthesis using polymerase-nucleotide conjugates. Nat Biotechnol. 36(7):645–650

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