An L-Aptamer-Displacement Assay for High-Throughput Screening of RNA-Targeted Small Molecule Antivirals

项目摘要/摘要 虽然绝大多数对抗严重急性呼吸综合征冠状病毒2(SARS-2)的抗病毒努力 CoV-2)专注于病毒的基本蛋白，新的证据表明保守的病毒RNA(VRNA)具有结构 元素是具有泛抗病毒活性潜力的引人注目的靶点。然而，尽管有这样的承诺， 使用类药物小分子选择性靶向RNA仍然具有挑战性。尤其是，用于 筛选抗RNA的小分子文库仍然不发达，没有充分解决 靶标专一性的中心问题。因此，以RNA为靶点的筛选往往无法产生有效的化合物。 建议的研究直接针对这一技术差距，通过开发一种新的靶向RNA L-镜象L核酸适体筛选技术。公安部此前认定L-- 适体可以进化成与天然D-RNA结构结合，包括SARS-CoV-2 vRNAs，具有高亲和力和 选择性。他现在建议将L适配子开发成RNA特异性的竞争置换探针 识别具有相似性质的小分子。核酸适配子的普遍用途 利用L适配子独特的核糖核酸结合特性，建立了L适配子置换分析方法。 比目前的以RNA为中心的筛选技术有几个优点，假设是为了促进 发现具有前所未有的RNA结合能力的小分子。 PI已经制备了针对SARS-CoV-2保守RNA元件的L适配子 基因组，这将被开发成一种生化分析，配对竞争置换的L- 来自vRNA靶的适体，带有光学读出(目标1)。使用这种测试，PI将启动一个高- 吞吐量筛选，以确定针对相应vRNA的有效配体。最有希望的线索 将评估化合物对SARS-CoV-2感染细胞的抗病毒活性(目标2)。并行的努力将 负责产生针对额外SARS-CoV-2RNA结构的L适体(目标3)，这将是 穿梭在同一条管道上。 该项目的成功完成将标志着RNA靶向药物发现领域的重大进展。 虽然抗击SARS-CoV-2是眼前的目标，但这里开发的技术很容易适应 瞄准任何RNA病毒。通过靶向β冠状病毒中保守的基本核糖核酸结构，猪 设想这种方法将允许鉴定具有广谱活性的抗病毒化合物， 可能会迅速转向应对未来的疫情。

Project Summary/Abstract While the vast majority of antiviral efforts to combat severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) focus on essential viral proteins, emerging evidence shows that conserved viral RNA (vRNA) structural elements are compelling targets with the potential for pan-antiviral activity. Despite this promise, however, selective targeting of RNA using drug-like small molecules remains challenging. In particular, methodologies for screening small molecule libraries against RNA remain underdeveloped, and do not adequately address the central problem of target specificity. As a result, RNA-targeted screens often fail to yield efficacious compounds. The proposed study takes direct aim at this technological gap through the development of a novel RNA-targeted screening technology using L-aptamers composed of mirror-image L-DNA. The PI previously established that L- aptamers can be evolved to bind native D-RNA structures, including SARS-CoV-2 vRNAs, with high affinity and selectivity. He now proposes to develop L-aptamers into RNA-specific competitive displacement probes for identifying small molecules with analogous properties. The general utility of nucleic acid aptamers, combined with the unique RNA-binding properties of L-aptamers, impart the proposed L-aptamer-displacement assay with several advantages over current RNA-centric screening technologies, and is hypothesized to facilitate the discovery of small molecules with unprecedented RNA-binding capabilities. The PI has already prepared an L-aptamer targeting a conserved RNA element with the SARS-CoV-2 genome, which will be developed into a biochemical assay that couples competitive displacement of the L- aptamer from the vRNA target with an optical readout (Aim 1). Using this assay, the PI will initiate a high- throughput screen to identify potent ligands targeting the corresponding vRNA. The most promising lead compounds will be evaluated for antiviral activity against SARS-CoV-2 infected cells (Aim 2). Parallel efforts will be undertaken to generate L-aptamers against additional SARS-CoV-2 RNA structures (Aim 3), which will be shuttled through this same pipeline. Successful completion of this project will signify a major advance in the area of RNA-targeted drug discovery. While combatting SARS-CoV-2 is the immediate goal, technologies developed herein are readily adaptable to target any RNA virus. By targeting essential RNA structures that are conserved across β-coronaviruses, the PI envision that this approach will allow for identification of antiviral compounds with broad-spectrum activity that might quickly pivot to address future outbreaks.