Engineering a denaturant-resistant polymerase for direct nucleic acid diagnostics

设计用于直接核酸诊断的抗变性聚合酶

• 批准号: 10308721
• 负责人: Barry Ryan Lutz
• 金额: $ 18.46万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308721
• 关键词: Address; Automation; Biological; Biological Assay; Biological Models; Biophysics; Buffers; Cells; Chemicals; Clinic; Clinical; Communicable Diseases; Complex; Coronavirus; Cytolysis; DNA; Detection; Development; Diagnosis; Diagnostic; Directed Molecular Evolution; Disease Outbreaks; Drops; Ebola; Engineering; Environment; Enzymes; Equipment; Excision; Exposure to; Genetic Diseases; Genome; Goals; Guidelines; HIV; HIV-1; Hepatitis Viruses; Human; Human immunodeficiency virus test; Influenza; Laboratories; Lateral; Libraries; Liquid substance; Measles; Mediating; Methods; Microfluidics; Modeling; Mutagenesis; Mutation; Nucleic Acid Amplification Tests; Nucleic Acids; Patients; Peptide Hydrolases; Plasma; Poliomyelitis; Polymerase; Preparation; Procedures; Proteins; Public Health; RNA; RNA Viruses; RNA purification; Rabies; Resistance; Resolution; Resource-limited setting; Resources; Reverse Transcription; Ribonucleases; Robotics; Sampling; Structure; Testing; Time; Tube; Variant; Virion; Virus; Vision; Work; amplification detection; cancer type; communicable disease diagnosis; cost; design; detection method; disease diagnostic; guanidine thiocyanate; health care settings; human tissue; inhibitor; insight; instrument; instrumentation; isothermal amplification; low income country; macromolecule; molecular dynamics; mutant; mutation screening; nucleic acid purification; point of care; point-of-care diagnostics; pressure; rational design; self testing; stem; tool; viral RNA

Project Abstract Nucleic acid amplification tests (NAATs) are powerful tools for infectious disease diagnostics. While NAATs are routinely used in the clinic, their use in point-of-care (POC) contexts is constrained by complex procedures needed to extract and purify nucleic acids from patient samples. This is especially troublesome for RNA targets, due to the fragility of RNA and abundance of RNA-degrading ribonucleases in samples and the environment. Nevertheless, RNA viruses are key targets for POC diagnostics, as they are abundant in low- resource settings (e.g. low-income countries, where roughly 20 million HIV patients live) or in settings where fast turnaround and patient self-testing are useful (e.g. outbreaks of airborne viruses such as influenza and coronavirus). Thus, simpler approaches to RNA sample preparation are needed in POC-NAAT contexts. Much of the complexity of NAAT sample preparation stems from the paradoxical need to add chaotropes to extract nucleic acids, then remove chaotropes before amplification. Chaotropes, such as guanidinium thiocyanate (GuSCN), are chemical denaturants that disrupt the structure of biological macromolecules. They are used in NAAT sample preparation to lyse target virions/cells and denature inhibitors, such as ribonucleases and proteases. Chaotropes are effective, but also inhibit polymerase activity, so they must be removed before amplification. Many POC adaptations of NAAT workflows involve robotic or microfluidic automation of chaotrope addition and removal, but still require specialized equipment and/or laboratory resources. We will address the POC-NAAT sample preparation bottleneck in a different way: instead of automating chaotrope removal, we will eliminate the need for it. We propose to engineer a polymerase to be “chaostable”, or active in a chaotropic amplification buffer, enabling simultaneous extraction, amplification, and detection of viral RNA targets in a single tube. To realize this vision, we propose three specific aims, using a starting polymerase previously developed by our lab and HIV-1 as a model RNA target. Aim 1: Develop a chaostable polymerase via compartmentalized self-replication in chaotropic conditions. We will use a high-throughput directed evolution approach to develop a polymerase that retains activity in 3M GuSCN (the minimum recommended concentration for RNA extraction buffers). Aim 2: Investigate mechanisms of polymerase chaotrope resistance via deep mutational scanning and molecular dynamics simulations. We will use deep mutational scanning and molecular dynamics simulations to study interactions of GuSCN on our polymerase and identify rational design methods for GuSCN resistance. Aim 3: Incorporate chaostable polymerase into a proof-of-concept HIV diagnostic that performs sample lysis, RT-LAMP amplification, and colorimetric detection in a single tube. Using the best-performing chaostable polymerases developed in Aims 1 and 2, we will design a single-tube RT-LAMP assay for HIV, and test it against pure HIV RNA and HIV patient plasma samples.

项目摘要 核酸扩增试验（NAAT）是传染病诊断的有力工具。虽然NAAT 常规用于临床，但其在床旁（POC）环境中的使用受到复杂程序的限制 需要从患者样本中提取和纯化核酸。这对RNA来说尤其麻烦 由于RNA的脆弱性和样品中大量的RNA降解核糖核酸酶， 环境尽管如此，RNA病毒是POC诊断的关键靶标，因为它们在低- 资源环境（例如，低收入国家，约有2000万艾滋病毒患者）或 快速周转和患者自我检测是有用的（例如，流感等空气传播病毒的爆发， 冠状病毒）。因此，在POC-NAAT背景下需要更简单的RNA样品制备方法。 NAAT样品制备的大部分复杂性源于向样品中加入离液剂的矛盾需要。 提取核酸，然后在扩增前除去离液剂。离液剂，如胍盐 硫氰酸盐（GuSCN）是破坏生物大分子结构的化学变性剂。他们 在NAAT样品制备中用于裂解靶病毒体/细胞和变性抑制剂，如核糖核酸酶 和蛋白酶。离液剂是有效的，但也会抑制聚合酶的活性，因此必须在 放大NAAT工作流程的许多POC适应涉及机器人或微流体自动化， 离液剂的添加和去除，但仍然需要专门的设备和/或实验室资源。 我们将以不同的方式解决POC-NAAT样品制备瓶颈： 我们建议设计一种聚合酶， “离液稳定的”，或在离液扩增缓冲液中有活性，能够同时提取， 扩增和检测单个管中的病毒RNA靶。为了实现这一愿景，我们提出三点建议。 具体目标，使用我们实验室先前开发的起始聚合酶和HIV-1作为模型RNA靶标。 目的1：通过离液序列中的区室化自我复制开发一种离液聚合酶。 条件我们将使用高通量定向进化方法来开发一种聚合酶， 3 M GuSCN中的活性（RNA提取缓冲液的最低推荐浓度）。 目的2：通过深度突变扫描和DNA测序研究聚合酶离液剂抗性的机制。 分子动力学模拟我们将使用深度突变扫描和分子动力学模拟 研究GuSCN对我们的聚合酶的相互作用，并确定GuSCN抗性的合理设计方法。 目的3：将混沌聚合酶纳入概念验证的HIV诊断中， 裂解、RT-LAMP扩增和比色检测。使用性能最好的 利用在目标1和2中开发的超稳定聚合酶，我们将设计用于HIV的单管RT-LAMP测定， 将其与纯HIV RNA和HIV患者血浆样本进行测试。