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

长而准确的合成DNA序列是未来生命科学和医疗保健的推动者，也是雄心勃勃的合成生物学家设计生物体的普遍工具。然而，制备时间、成本、精确度和通过当今标准化学合成方法获得的碱基长度是限制合成DNA在新型文库构建、合成基因和整个合成基因组的组装中的使用的因素。新兴的基于酶的DNA合成有望克服事实上的化学DNA合成方法的局限性。我的研究愿景是证明基于酶促DNA合成的多千碱基DNA的高速、无错误、从头合成的可行性，超越当前寡核苷酸合成和基因组装方法的局限性。我的研究计划的新奇是光电微反应器系统的发展，直接读出和控制末端脱氧核苷酸转移酶（TdT）酶活性的序列定义的DNA从头合成。这种方法将联合收割机结合高保真的生物化学TdT为基础的DNA合成与国家的最先进的光等离子体单分子传感和光电/流体控制的单分子反应。光电微反应器将允许我通过在真实的时间内控制TdT酶的每个反应步骤来控制DNA合成，并且写入序列定义的DNA，而不需要对TdT酶的核苷酸或功能突变进行任何化学改变，从而实现比当前方法更高的合成速度和准确性。目的是合成任意DNA，接近野生型TdT周转率200碱基/分钟，错误率为10-4/碱基。实现这些指标将为目前的DNA合成能力提供一个台阶式的变化，将目前每分钟2-3个碱基的掺入率提高100倍，并将目前的错误率提高10倍。我的研究计划将为开发多酶DNA作家奠定基础，并为实施新的超精确化学合成方法铺平道路。

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.