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Docking and Activity of DNA Polymerase on Solid-State Nanopores
ACS Sensors ( IF 8.2 ) Pub Date : 2022-05-10 , DOI: 10.1021/acssensors.2c00216
Shiyu Li 1 , Shuangshuang Zeng 1 , Chenyu Wen 1 , Zhen Zhang 1 , Klas Hjort 2 , Shi-Li Zhang 1
Affiliation  

Integration of motor enzymes with biological nanopores has enabled commercial DNA sequencing technology; yet studies of the similar principle applying to solid-state nanopores are limited. Here, we demonstrate the real-life monitoring of phi29 DNA polymerase (DNAP) docking onto truncated-pyramidal nanopore (TPP) arrays through both electrical and optical readout. To achieve effective docking, atomic layer deposition of hafnium oxide is employed to reduce the narrowest pore opening size of original silicon (Si) TPPs to sub-10 nm. On a single TPP with pore opening size comparable to DNAP, ionic current measurements show that a polymerase–DNA complex can temporally dock onto the TPP with a certain docking orientation, while the majority become translocation events. On 5-by-5 TPP arrays, a label-free optical detection method using Ca2+ sensitive dye, are employed to detect the docking dynamics of DNAP. The results show that this label-free detection strategy is capable of accessing the docking events of DNAP on TPP arrays. Finally, we examine the activity of docked DNAP by performing on-site rolling circle amplification to synthesize single-stranded DNA (ssDNA), which serves as a proof-of-concept demonstration of utilizing this docking scheme for emerging nanopore sensing applications.

中文翻译:

DNA聚合酶在固态纳米孔上的对接和活性

运动酶与生物纳米孔的整合使商业 DNA 测序技术成为可能;然而,适用于固态纳米孔的类似原理的研究是有限的。在这里,我们展示了通过电子和光学读数对 phi29 DNA 聚合酶 (DNAP) 对接到截短锥体纳米孔 (TPP) 阵列的实际监测。为了实现有效对接,采用氧化铪的原子层沉积将原始硅 (Si) TPP 的最窄孔径减小到 10 nm 以下。在具有与 DNAP 相当的孔径大小的单个 TPP 上,离子电流测量表明聚合酶-DNA 复合物可以以一定的对接方向暂时对接到 TPP 上,而大多数成为易位事件。在 5×5 TPP 阵列上,一种使用 Ca 的无标记光学检测方法2+敏感染料,用于检测DNAP的对接动力学。结果表明,这种无标记检测策略能够访问TPP阵列上DNAP的对接事件。最后,我们通过进行现场滚环扩增以合成单链 DNA (ssDNA) 来检查对接 DNAP 的活性,这可作为将这种对接方案用于新兴纳米孔传感应用的概念验证演示。
更新日期:2022-05-10
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