A biophysical assay targeting SARS CoV-2 RNA

项目总结 世界正沉浸在一场仅次于1918-1919年西班牙流感大流行的健康危机中。区别在于 在今天的危机和一个世纪前的危机之间，我们拥有先进的通信技术 以便全球各地的大量人口意识到风险并采取适当的措施 预防措施，如使用隔离、隔离、社交距离、口罩和洗手。 政府已经关闭了学校，并宣布大型社交集会为非法。医疗服务有了很大的改善， 而受影响最严重的人在新冠肺炎无法治愈的情况下接受了对症治疗。这个 必须在多个方面处理问题，如疫苗开发、药物开发和新技术， 减轻病毒影响的化验。拟议的项目意义重大，因为它提出了一种新颖的 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 病毒，将来可以用来产生RNA特异性的抗病毒化合物。 核酸是药物设计的很有前途的途径，既是治疗药物，也是靶点。在这里，我们提出一种 用于识别一类新的RNA特异性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成分，我们建议一种 生物物理筛选试验是鉴定这类配体的第一步。第一，如《具体目标》中所述 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 鉴定一种RNA特异性荧光探针。然后，优化后的探头将用于开发高通量的 发现与该RNA的纳米分子结合的筛选试验。然后，RNA特异的高亲和力结合物将 与序列特异的RNA结合配体结合，以验证该方法的发展及其实用性。这个 作用机制将通过抑制萤火虫荧光素酶的翻译在报告基因测试中得到证实 (具体目标2)。虽然这个应用程序的重点，作为必要的第一步，是在开发和 对SARS-CoV-2潜在RNA结构的生物物理测试的验证，成功地验证了 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 除了为科学界提供模板外，还在第二阶段分析和开发潜在的线索 将筛选技术用于他们自己的发现平台。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.