磷酰胺
计算生物学
末端脱氧核苷酸转移酶
寡核苷酸
DNA合成
化学
生物
组合化学
酶
适体
计算机科学
DNA
生物化学
分子生物学
细胞凋亡
标记法
作者
Michael A. Jensen,Ronald W. Davis
出处
期刊:Biochemistry
[American Chemical Society]
日期:2018-03-13
卷期号:57 (12): 1821-1832
被引量:51
标识
DOI:10.1021/acs.biochem.7b00937
摘要
There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is comprised of additions > n+1 and deletions. By tapping into the physical and biochemical properties of TdT, we strive to further elucidate its mercurial behavior and offer ways to better optimize TiEOS for production-grade oligonucleotide synthesis.
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