Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597.
NUS Synthetic Biology for Clinical and Technological Innovation, 14 Medical Drive, Singapore 117599.
ACS Synth Biol. 2020 Jul 17;9(7):1725-1735. doi: 10.1021/acssynbio.0c00078. Epub 2020 Jun 23.
Terminal deoxynucleotidyl transferase (TdT) catalyzes template free incorporation of arbitrary nucleotides onto single-stranded DNA. Due to this unique feature, TdT is widely used in biotechnology and clinical applications. One particularly tantalizing use is the synthesis of long DNA molecules by TdT-mediated iterative incorporation of a 3' reversibly blocked nucleotide, followed by deblocking. However, wild-type (WT) TdT is not optimized for the incorporation of 3' modified nucleotides, and TdT engineering is hampered by the fact that TdT is marginally stable and only present in mesophilic organisms. We sought to first evolve a thermostable TdT variant to serve as backbone for subsequent evolution to enable efficient incorporation of 3'-modified nucleotides. A thermostable variant would be a good starting point for such an effort, as evolution to incorporate bulky modified nucleotides generally results in lowered stability. In addition, a thermostable TdT would also be useful when blunt dsDNA is a substrate as higher temperature could be used to melt dsDNA. Here, we developed an assay to identify thermostable TdT variants. After screening about 10 000 TdT mutants, we identified a variant, named TdT3-2, that is 10 °C more thermostable than WT TdT, while preserving the catalytic properties of the WT enzyme.
末端脱氧核苷酸转移酶（TdT）催化在单链 DNA 上无模板掺入任意核苷酸。由于这一独特的特性，TdT 在生物技术和临床应用中得到了广泛的应用。一个特别诱人的用途是通过 TdT 介导的 3'可逆封锁核苷酸的迭代掺入，然后进行去封锁，来合成长的 DNA 分子。然而，野生型（WT）TdT 不适于掺入 3'修饰核苷酸，并且 TdT 的工程受到 TdT 稳定性差和仅存在于嗜温生物中的事实的阻碍。我们首先寻求进化出一种耐热 TdT 变体，作为随后进化的骨干，以实现 3'-修饰核苷酸的有效掺入。耐热变体将是此类努力的一个很好的起点，因为掺入大体积修饰核苷酸的进化通常会导致稳定性降低。此外，当钝末端 dsDNA 作为底物时，耐热 TdT 也将非常有用，因为可以使用更高的温度来使 dsDNA 融化。在这里，我们开发了一种鉴定耐热 TdT 变体的测定法。在筛选了大约 10000 个 TdT 突变体后，我们鉴定出一个名为 TdT3-2 的变体，比 WT TdT 耐热 10°C，同时保留了 WT 酶的催化特性。
