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Sci Adv. 2023 Jul 7;9(27):eadi0263. doi: 10.1126/sciadv.adi0263.
Enzymatic DNA synthesis (EDS) is a promising benchtop and user-friendly method of nucleic acid synthesis that, instead of solvents and phosphoramidites, uses mild aqueous conditions and enzymes. For applications such as protein engineering and spatial transcriptomics that require either oligo pools or arrays with high sequence diversity, the EDS method needs to be adapted and certain steps in the synthesis process spatially decoupled. Here, we have used a synthesis cycle comprising a first step of site-specific silicon microelectromechanical system inkjet dispensing of terminal deoxynucleotidyl transferase enzyme and 3' blocked nucleotide, and a second step of bulk slide washing to remove the 3' blocking group. By repeating the cycle on a substrate with an immobilized DNA primer, we show that microscale spatial control of nucleic acid sequence and length is possible, which, here, are assayed by hybridization and gel electrophoresis. This work is distinctive for enzymatically synthesizing DNA in a highly parallel manner with single base control.
酶促 DNA 合成（EDS）是一种很有前途的、适用于实验室的、用户友好型核酸合成方法，它使用温和的水性条件和酶代替溶剂和磷酰胺。对于需要寡核苷酸池或具有高序列多样性的阵列的蛋白质工程和空间转录组学等应用，EDS 方法需要进行适配，并且需要将合成过程中的某些步骤在空间上解耦。在这里，我们使用了一个合成周期，包括第一步骤为末端脱氧核苷酸转移酶和 3' 封闭核苷酸的硅微机电系统喷墨分配的特异性位点，以及第二步骤为批量滑动洗涤以去除 3' 封闭基团。通过在固定有 DNA 引物的基底上重复该循环，我们证明了核酸序列和长度的微尺度空间控制是可行的，在这里，通过杂交和凝胶电泳进行了检测。这项工作的独特之处在于以单碱基控制的方式高度平行地酶促合成 DNA。
