High-speed de novo DNA writer by single-molecule control of TdT enzyme

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Long, accurate sequences of synthetic DNA are an enabler for future life science and healthcare and a ubiquitous tool for ambitious synthetic biologists to engineer organisms. However, preparation time, cost, accuracy and base length attained by today's standard chemical synthesis methods are the factors that limit the use of synthetic DNA in novel library construction, synthetic genes and the assembly of entire synthetic genomes. Emerging DNA synthesis based on enzymes holds promise for overcoming the limitations of the de facto chemical DNA synthesis methods. My research vision is to demonstrate the feasibility of high-speed, error-free, de novo synthesis of multi-kilo base DNA based on enzymatic DNA synthesis surpassing the limitations of current oligo synthesis and gene assembly methods. The novelty of my research programme is the development of an optoelectronic microreactor system which directly reads out and controls Terminal deoxynucleotidyl Transferase (TdT) enzyme activity for sequence-defined DNA de novo synthesis. This approach will combine the high-fidelity of biochemical TdT-based DNA synthesis with state-of-the-art optoplasmonic single-molecule sensing and the optoelectronic/fluidic control of the single-molecule reaction. The optoelectronic microreactor will allow me to control DNA synthesis by controlling each reaction step of the TdT enzyme in real time and to write sequence-defined DNA without requiring any chemical alteration of the nucleotides or functional mutations of the TdT enzyme thus achieving an increase in synthesis speed and accuracy over the current methods. The aim is to synthesize arbitrary DNA approaching the wild-type TdT turnover rate of 200 bases/minute with an error rate of 10-4 per base. Achieving these metrics will provide a step-change for the current DNA synthesis capabilities, improving the current incorporation rates of 2-3 bases per minute 100-fold and improving the current error rates 10-fold. My research programme will lay the groundwork for developing multi-kilobase DNA writers and pave the way for implementing novel ultra-precise chemical synthesis methods.