Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

Wanchao Yin; Chunyou Mao; Xiaodong Luan; Dan-Dan Shen; Qingya Shen; Haixia Su; Xiaoxi Wang; Fulai Zhou; Wenfeng Zhao; Minqi Gao; Shenghai Chang; Yuan-Chao Xie; Guanghui Tian; He-Wei Jiang; Sheng-Ce Tao; Jingshan Shen; Yi Jiang; Hualiang Jiang; Yechun Xu; Shuyang Zhang; Yan Zhang; H. Eric Xu

The purified SARS-CoV-2 RdRp complex from insect cell at final concentration of 1 μM was incubated with 3.0 μM dsRNA and 10 mM ATP in the presence of 1.14 U/μl RNase inhibitor in Reaction buffer containing 20 mM Tris pH8.0, 10 mM KCl, 6 mM MgCl2, 0.01% Triton-X100, 1 . CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.08.032763 doi: bioRxiv preprint mM DTT, which were prepared with DEPC-treated water.The total reaction volume was 20 μl. After incubation for 0 min, 5 min, 10 min, 20 min, 30 min, 45 min, and 60 min at 37°C water bath, 40 μl quench buffer (94% formamide, 30 mM EDTA, prepare with 2x Reaction buffer) was added to stop the reaction. A sample of 18 μl of reaction was mixed with 2 μl 10x DNA loading buffer and half of the sample (10 μl) was loaded onto a 20% Urea-PAGE denatured gel and run at 120V for 1h. Image via Tanon-5200 Multi Fluorescence Imager.