Abstract：
Enzymatic DNA writing technologies based on the template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) have the potential to advance DNA information storage. TdT is unique in its ability to synthesize single-stranded DNA de novo but has limitations, including catalytic inhibition by ribonucleotide presence and slower incorporation rates compared to replicative polymerases. We anticipate that protein engineering can improve, modulate, and tailor the enzyme\'s properties, but there is limited information on TdT sequence-structure-function relationships to facilitate rational approaches. Therefore, we developed an easily modifiable screening assay that can measure the TdT activity in high-throughput to evaluate large TdT mutant libraries. We demonstrated the assay\'s capabilities by engineering TdT mutants that exhibit both improved catalytic efficiency and improved activity in the presence of an inhibitor. We screened for and identified TdT variants with greater catalytic efficiency in both selectively incorporating deoxyribonucleotides and in the presence of deoxyribonucleotide/ribonucleotide mixes. Using this information from the screening assay, we rationally engineered other TdT homologues with the same properties. The emulsion-based assay we developed is, to the best of our knowledge, the first high-throughput screening assay that can measure TdT activity quantitatively and without the need for protein purification.
摘要：
基于模板非依赖性DNA聚合酶末端脱氧核苷酸转移酶（TdT）的酶促DNA写入技术具有促进DNA信息存储的潜力。TdT在从头合成单链DNA的能力上是独一无二的，但有局限性，包括核糖核苷酸存在的催化抑制和与复制聚合酶相比更慢的掺入率。我们预计蛋白质工程可以改善，调制,调整酶的特性，但是关于TdT序列-结构-功能关系的信息有限，无法促进合理的方法。因此,我们开发了一种易于修改的筛选试验,该试验可以高通量测量TdT活性,以评估大型TdT突变文库.我们通过改造TdT突变体证明了该测定法的能力,所述突变体在抑制剂存在下表现出提高的催化效率和提高的活性。我们筛选并鉴定了在选择性掺入脱氧核糖核苷酸和存在脱氧核糖核苷酸/核糖核苷酸混合物的情况下具有更大催化效率的TdT变体。利用这些来自筛选试验的信息，我们合理地设计了其他具有相同性质的TdT同系物。我们开发的基于乳液的检测方法是，据我们所知,这是第一个可以定量测量TdT活性且无需蛋白质纯化的高通量筛选测定法。
