State Key Laboratory for Advanced Metals and Materials, School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, P. R. China.
Beijing National Laboratory for Molecular Sciences, National Biomedical Imaging Centre, College of Chemistry and Molecular Engineering, Peking University, 292 Chengfu Road, Haidian District, Beijing, 100871, P. R. China.
Nat Commun. 2023 Feb 1;14(1):552. doi: 10.1038/s41467-023-36278-6.
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.
RNA 的降解过程对于保证生物体内遗传信息的高保真翻译至关重要。然而，实时观察单碱基降解过程并在单酶水平上破译降解机制仍然是巨大的挑战。在这里，我们提出了一种基于硅纳米线场效应晶体管的可靠原位单 PNPase 分子动态电探测器，具有超高的时间分辨率。这些器件能够实现实时和无标记的 RNA 类似物降解单碱基分辨率监测，包括 RNA 类似物结合、单核苷酸水解和单碱基移动。我们发现了酶（靠近活性位点）与核苷的结合事件，进一步了解了 RNA 降解机制。通过对独立读取的系统分析，在单个测试过程中实现了约 80%的 RNA 核苷测序准确率。这一概念验证为开发高通量检测技术提供了一个互补金属氧化物半导体 (CMOS) 兼容的平台，用于对机制进行探索和单分子测序。
