Enzymatic Synthesis of Designer DNA Using Cyclic Reversible Termination and a Universal Template
Enzymatic Synthesis of Designer DNA Using Cyclic Reversible Termination and a Universal Template
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DOI：
10.1021/acssynbio.9b00315
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发表时间：
2020-02-01
影响因子：
4.7
通讯作者：
Zhou, Wei
中科院分区：
文献类型：
--
作者：
Hoff, Kendall;Halpain, Michelle;Zhou, Wei
尽管在寡核苷酸的长度和产率、时间限制和产生危险废物方面存在重大限制，但磷酸酰胺化学仍然是DNA合成的工业标准。在这里，我们展示了DNA聚合酶和逆转录酶在固体表面上合成单链寡核苷酸。我们报道了表面结合寡核苷酸的扩展，使瞬时杂交少至两个碱基到邻近链。当多种杂交结构成为可能时，每个模板都有不同的碱基，DNA聚合酶或逆转录酶可以用任何互补碱基延伸寡核苷酸。因此，新合成片段的序列可以通过仅将所需的碱作为底物添加到反应溶液中来控制。我们使用这种酶的方法合成了一个20碱基的寡核苷酸，通过两步循环可逆终止过程加入可逆终止dNTPs，校正后的逐步效率超过98%。在我们的方法中，作为引物和模板的新生DNA链通过聚合酶控制的3‘可逆阻断核苷酸的顺序添加以及随后的3’ capping基团的切割进行扩展。该工艺可以在传统的磷酸酰胺方法所不允许的环境中合成寡核苷酸，消除了对危险化学品的需求，具有提供更快和更高收率结果的潜力，并且在具有游离3'端的固体载体上合成DNA。
Phosphoramidite chemistry remains the industry standard for DNA synthesis despite significant limitations on the length and yield of the oligonucleotide, time restrictions, and hazardous waste production. Herein, we demonstrate the synthesis of single-stranded oligos on a solid surface by DNA polymerases and reverse transcriptases. We report the extension of surface-bound oligonucleotides enabled by transient hybridization of as few as two bases to a neighboring strand. When multiple hybridization structures are possible, each templating a different base, a DNA polymerase or reverse transcriptase can extend the oligonucleotide with any of the complementary bases. Therefore, the sequence of the newly synthesized fragment can be controlled by adding only the desired base as a substrate to the reaction solution. We used this enzymatic approach to synthesize a 20 base oligonucleotide by incorporating reversible terminator dNTPs through a two-step cyclic reversible termination process with a corrected stepwise efficiency over 98%. In our approach, a nascent DNA strand that serves as both primer and template extended through polymerase-controlled sequential addition of 3'-reversibly blocked nucleotides followed by subsequent cleavage of the 3'-capping group. This process enables oligonucleotide synthesis in an environment not permitted by traditional phosphoramidite methods, eliminates the need for hazardous chemicals, has the potential to provide faster and higher yield results, and synthesizes DNA on a solid support with a free 3' end.