Single-exonuclease nanocircuits reveal the RNA degradation dynamics of PNPase and demonstrate potential for RNA sequencing

Zhiheng Yang, Wenzhe Liu, Lihua Zhao, Dongbao Yin, Jianfei Feng, Lidong Li () and Xuefeng Guo ()
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Zhiheng Yang: University of Science and Technology Beijing
Wenzhe Liu: Peking University, 292 Chengfu Road, Haidian District
Lihua Zhao: Peking University, 292 Chengfu Road, Haidian District
Dongbao Yin: University of Science and Technology Beijing
Jianfei Feng: Peking University, 292 Chengfu Road, Haidian District
Lidong Li: University of Science and Technology Beijing
Xuefeng Guo: Peking University, 292 Chengfu Road, Haidian District

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: Abstract The degradation process of RNA is decisive in guaranteeing high-fidelity translation of genetic information in living organisms. However, visualizing the single-base degradation process in real time and deciphering the degradation mechanism at the single-enzyme level remain formidable challenges. Here, we present a reliable in-situ single-PNPase-molecule dynamic electrical detector based on silicon nanowire field-effect transistors with ultra-high temporal resolution. These devices are capable of realizing real-time and label-free monitoring of RNA analog degradation with single-base resolution, including RNA analog binding, single-nucleotide hydrolysis, and single-base movement. We discover a binding event of the enzyme (near the active site) with the nucleoside, offering a further understanding of the RNA degradation mechanism. Relying on systematic analyses of independent reads, approximately 80% accuracy in RNA nucleoside sequencing is achieved in a single testing process. This proof-of-concept sets up a Complementary Metal Oxide Semiconductor (CMOS)-compatible playground for the development of high-throughput detection technologies toward mechanistic exploration and single-molecule sequencing.

Date: 2023
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DOI: 10.1038/s41467-023-36278-6

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