Harnessing DNA as a high-density storage medium for information storage and molecular recording of signals has been of increasing interest in the biotechnology field. Recently, progress in enzymatic DNA synthesis, DNA digital data storage, and DNA-based molecular recording has been made by leveraging the activity of the template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT). TdT adds deoxyribonucleotides to the 3′ end of single-stranded DNA, generating random sequences of single-stranded DNA. TdT can use several divalent cations for its enzymatic activity and exhibits shifts in deoxyribonucleotide incorporation frequencies in response to changes in its reaction environment. However, there is limited understanding of sequence-structure-function relationships regarding these properties, which in turn limits our ability to modulate TdT to further advance TdT-based tools. Most TdT literature to-date explores the activity of murine, bovine or human TdTs; studies probing TdT sequence and structure largely focus on strictly conserved residues that are functionally critical to TdT activity. Here, we explore non-conserved TdT sequence space by surveying the natural diversity of TdT. We characterize a diverse set of TdT homologs from different organisms and identify several TdT residues/regions that confer differences in TdT behavior between homologs. The observations in this study can design rules for targeted TdT libraries, in tandem with a screening assay, to modulate TdT properties. Moreover, the data can be useful in guiding further studies of potential residues of interest. Overall, we characterize TdTs that have not been previously studied in the literature, and we provide new insights into TdT sequence-function relationships.

中文翻译：

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阐明不依赖模板的聚合酶中的序列-功能关系，以实现新型 DNA 记录应用


利用 DNA 作为信息存储和信号分子记录的高密度存储介质在生物技术领域越来越受到关注。最近，通过利用不依赖模板的 DNA 聚合酶末端脱氧核苷酸转移酶 （TdT） 的活性，在酶促 DNA 合成、DNA 数字数据存储和基于 DNA 的分子记录方面取得了进展。TdT 将脱氧核糖核苷酸添加到单链 DNA 的 3' 端，生成单链 DNA 的随机序列。TdT 可以使用多种二价阳离子来实现其酶活性，并表现出响应其反应环境变化的脱氧核糖核苷酸掺入频率的变化。然而，对这些特性的序列-结构-功能关系的理解有限，这反过来又限制了我们调节 TdT 以进一步推进基于 TdT 的工具的能力。迄今为止，大多数 TdT 文献探讨了小鼠、牛或人 TdT 的活性;探索 TdT 序列和结构的研究主要集中在对 TdT 活性功能至关重要的严格保守的残基上。在这里，我们通过调查 TdT 的自然多样性来探索非守恒 TdT 序列空间。我们表征了来自不同生物体的一组不同的 TdT 同源物，并确定了几个赋予同源物之间 TdT 行为差异的 TdT 残基/区域。本研究中的观察结果可以设计靶向 TdT 文库的规则，与筛选测定相结合，以调节 TdT 特性。此外，这些数据可用于指导对潜在目标残基的进一步研究。总体而言，我们表征了以前在文献中未研究过的 TdT，并为 TdT 序列-功能关系提供了新的见解。