Model-guided design of RNA stabilizing elements for improved coronavirus diagnostics

项目摘要 检测SARS-CoV-2冠状病毒的快速、廉价和敏感的方法是有用的 在遏制新冠肺炎的传播方面。目前，呼吸道样本的核酸检测使用 RT-PCR是诊断急性白血病患者的主要方法，也是最常用的方法。 感染的阶段。然而，这种标准方法需要特殊的 设备和训练有素的人员，因此成为满足紧迫需求的瓶颈 对大规模筛选的需求。一系列基于RNA的新技术，包括Toehold 正在积极开发交换机和CRISPR/CAS系统，目的是实施 超灵敏、易于部署、利用酶和试剂的诊断测试 与传统的聚合酶链式反应流水线分离。这些方法的一个共同且关键的组成部分 是通过改造RNA分子来检测目标病毒序列。因此，他们的 在特异性和敏感性方面的表现已经受到FAST的严重阻碍 临床样本和AS中普遍存在的核糖核酸酶引起的RNA降解 生物分子试剂生产的副产品。 在这里，我们建议通过编程RNase来提高冠状病毒诊断性能 抗性转化为检测成分，进而提高了RNA的稳定性并增强了检测 敏感度和速度。我们将合理设计RNA5‘非编码区序列和由此产生的 MRNA、Toehold Switch RNA和CRISPR的二级结构引导RNA调节 它们对核糖核酸酶活性的抵抗力，从而定量地调整它们的稳定性。结果 从提出的未来工程研究将增加我们对 并提供了一种广泛适用的策略来改进冠状病毒检测 许多正在开发的技术的效率。Impact：一种被广泛研究的RNA 提高RNA稳定性的设计方案将补充现有的技术 开发以提高性能，并为基础设计策略提供 潜在的更广泛的适用性，例如克服基于mRNA的疫苗的稳定性障碍。 这项提议有三个具体目标：目标1：描述和示范5‘的作用 微调信使核糖核酸稳定性的二级结构；目标2：优化检测的传感核糖核酸 新冠肺炎；目标3：使用dtRNA增强样品和扩增RNA的稳定性，以改善 诊断。

Project Summary Fast, inexpensive, and sensitive methods to detect the SARS-CoV-2 coronavirus are instrumental in containing the spread of COVID-19. Currently, nucleic acid testing of respiratory samples using RT-PCR is the primary and most commonly used method for diagnosing patients in the acute phase of the infection. However, this standard approach suffers from the need for special equipment and well-trained personnel and hence has become a bottleneck to meet the urgent demand for large-scale screening. A range of new RNA-based technologies, including toehold switches and CRISPR/Cas systems, are being actively developed with the aim to implement diagnostic tests that are ultra-sensitive, easy to deploy, and make use of enzymes and reagents separate from the traditional PCR pipeline. One common and critical component of these methods is the engineering of RNA molecules to detect target viral sequences. Consequently, their performance in terms of specificity and sensitivity have been significantly hindered by the fast degradation of RNAs caused by the RNases ubiquitous in both clinical sample matrices and as byproducts of biomolecular reagent production. Here we propose to enhance coronavirus diagnostic performance by programming RNase resistance into assay components, in turn increasing RNA stability and enhancing test sensitivity and speed. We will rationally design RNA 5’ UTR sequences and the resulting secondary structures of mRNA, toehold switch RNA, and CRISPR guide RNAs to modulate their resistance to RNase activities and hence quantitatively tune their stability. Results from the proposed forward engineering studies will increase our understanding and control of RNA dynamics and provide a widely applicable strategy to improve coronavirus detection efficiencies of many technologies under development. Impact: A comprehensively studied RNA design scheme to improve RNA stability will be complementary to current technologies under development to give them a boost in performance, and provide underlying design strategies with potential broader applicability, such as overcoming the stability barrier for mRNA-based vaccines. There are three specific aims in this proposal: Aim 1: Characterize and model the role of 5’ secondary structures in fine-tuning mRNA stability; Aim 2: Optimize sensing RNAs for detection of COVID-19; Aim 3: Use dtRNAs to enhance sample and amplified RNA stability for improved diagnostics.