SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process including the nonstructural protein 16 (nsp16) which is an S-adenosyl-L-methionine (SAM)-dependent 2'-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in 384-well format with a Z′-Factor of 0.6, suitable for high throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, product of the reaction, SAH, and a common methyltransferase inhibitor, sinefungin using Isothermal Titration Calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high throughput method for screening nsp10-nsp16 complex for RNA-competitive inhibitors towards developing COVID-19 therapeutics.

中文翻译：

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高通量RNA置换分析用于筛选SARS-CoV-2 nsp10-nsp16复合物，以开发COVID-19的疗法

SARS-CoV-2是导致COVID-19的冠状病毒，它通过封盖其RNA逃避人类免疫系统。该过程保护病毒RNA，对于其复制至关重要。多种病毒蛋白都参与了该RNA封闭过程，包括非结构蛋白16（nsp16），它是S-腺苷-L-蛋氨酸（SAM）依赖性2'-O-甲基转移酶。当与另一种非结构蛋白nsp10复合时，Nsp16具有显着活性，nsp10在其稳定性和活性中起关键作用。在这里，我们报告以384孔格式的nsp10-nsp16复合物基于荧光偏振（FP）的RNA置换分析方法的发展，其Z'-因子为0.6，适用于高通量筛选。在此过程中，我们将nsp10-nsp16复合物纯化至高于95％的纯度，并确认了其与甲基供体SAM的结合，反应的产物SAH和常用的甲基转移酶抑制剂西氟芬净（等温滴定热量法（ITC））。通过筛选1124种药物样化合物的文库进一步验证了该测定法。该测定法提供了一种经济高效的高通量方法，可用于筛选nsp10-nsp16复合物的RNA竞争性抑制剂，以开发COVID-19治疗剂。