Docking and Activity of DNA Polymerase on Solid-State Nanopores.

Shiyu Li, Shuangshuang Zeng, Chenyu Wen, Zhen Zhang, Klas Hjort, Shi-Li Zhang

ACS sensors 2022 May 27

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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.

Citation

Shiyu Li, Shuangshuang Zeng, Chenyu Wen, Zhen Zhang, Klas Hjort, Shi-Li Zhang. Docking and Activity of DNA Polymerase on Solid-State Nanopores. ACS sensors. 2022 May 27;7(5):1476-1483