A biophysical assay targeting SARS CoV-2 RNA

项目摘要 世界正陷入一场健康危机，只有1918-1919年的西班牙流感大流行可与之媲美。的差异 当今的危机和一个世纪前的危机之间的区别在于我们拥有先进的通讯技术 使地球仪上的大量人口意识到风险，并能够采取适当的措施， 预防措施，如使用隔离、隔离、社交距离、口罩和洗手。 政府关闭了学校，禁止大型社交聚会。医疗保健有了很大的改善， 而那些受影响最严重的人在没有治愈COVID-19的情况下接受了对症治疗。的 必须从疫苗开发、药物开发和新技术等多个方面解决问题， 用于减轻病毒作用的测定。这个项目很重要，因为它提出了一个新的 SARS-CoV-2中一种独特但尚未开发的RNA结构的体外生物物理筛选方法 病毒，这可以在未来用于产生RNA特异性抗病毒化合物。 核酸是药物设计的有希望的途径，既作为治疗剂又作为靶点。在这里，我们提出一个 用于鉴定对RNA特异的新型配体的创新筛选方法 病毒RNA基因组中对病毒复制至关重要的元件，我们提出了一个 生物物理筛选测定作为鉴定此类配体的第一步。第一，具体目标 1，我们将表征一个模型核酸（RNA）结构域，该结构域将被合成、表征并用于 鉴定RNA特异性荧光探针。然后将优化的探针用于开发高通量的 筛选测定以发现该RNA的纳摩尔结合物。然后，RNA特异性高亲和力结合剂将 与序列特异性RNA结合配体组合，以验证测定开发及其实用性。的 将在报告基因测定中使用萤火虫荧光素酶翻译的抑制来证实作用机制 （具体目标2）。虽然这个应用程序的重点，作为所需的第一步，是在开发和 验证了SARS-CoV-2 RNA结构的生物物理分析，成功验证了 该方法将为发现和开发抑制SARS-CoV的先导化合物打开大门 在第二阶段的研究中。NUBAD及其科学家和合作者团队是唯一有资格执行这些任务的人。 除了为科学界提供一个模板外， to use the screening筛选technology技术for their其own拥有discovery发现platforms平台.该方法的成功还将使我们能够 将筛选技术扩展到SARS和其他RNA病毒中的其他RNA结构，并提供 筛选资源，为科学界提供服务。

PROJECT SUMMARY The world is immersed in a health crisis rivaled only by the Spanish flu pandemic of 1918-1919. The difference between the crisis of today and the crisis a century ago is that we have advanced communication technology greatly so that huge populations of people around the globe are aware of risks and can take appropriate precautions, such as the employment of quarantining, isolation, social distancing, and masks and handwashing. Governments have closed schools and outlawed large social gatherings. Medical care has greatly improved, and those most affected have received symptomatic treatment in the absence of a cure for CoVID-19. The problem must be dealt at multiple fronts, such as vaccine development, drug development and new technologies, assays for mitigating viral effects. The proposed project is significant because it proposes a novel in vitro biophysical screening assay for a unique and yet untapped RNA structure in SARS-CoV-2 virus, that can be used in the future to generate RNA specific antiviral compounds. Nucleic acids are promising avenues for drug design, both as therapeutics and as targets. Here we propose an innovative screening approach for identification of a novel class of ligands that are specific for an RNA element within the viral RNA genome that is vital for replication of the virus, and we propose a biophysical screening assay as a first step for identifying such ligands. First, as outlined in Specific Aim 1, we will characterize a model nucleic acid (RNA) domain that will be synthesized, characterized and used to identify a RNA specific fluorescent probe. The optimized probe will then be used for developing a high-throughput screening assay for discovery of nanomolar binders to this RNA. The RNA specific high-affinity binders will then be combined with sequence-specific RNA binding ligands to validate the assay development and its utility. The mechanism of action will be confirmed using inhibition of firefly luciferase translation in a reporter gene assay (Specific Aim 2). While the focus of this application, as the needed first step, is on the development and validation of the biophysical assay for the underlying SARS-CoV-2 RNA structures, a successful validation of the approach will open the doors for discovery and development of lead compounds for inhibition of SARS-CoV in Phase II studies. NUBAD and its team of scientists and collaborators is uniquely qualified to perform these assays and develop the potential leads in Phase II, in addition to providing a template for the scientific community to use the screening technology for their own discovery platforms. Success of the approach will also allow us to expand the screening technology to other RNA structures in SARS and other RNA viruses, and provide the screening resource as a service to the scientific community.