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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要核酸扩增测试 (NAAT) 是传染病诊断的强大工具。常规在诊所使用，但其在护理点 (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，这对于 RNA 来说尤其麻烦。由于 RNA 的脆弱性和样品中 RNA 降解核糖核酸酶的丰富性，然而，RNA 病毒是 POC 诊断的关键目标，因为它们在低浓度环境中含量丰富。资源环境（例如低收入国家，大约有 2000 万艾滋病毒患者居住）或环境快速周转和患者自我测试是有用的（例如，流感和流感等空气传播病毒的爆发）因此，在 POC-NAAT 环境中需要更简单的 RNA 样品制备方法。 NAAT 样品制备的大部分复杂性源于添加离液剂的矛盾需求提取核酸，然后在扩增前去除离液剂，例如胍盐。硫氰酸盐（GuSCN）是破坏生物大分子结构的化学变性剂。用于 NAAT 样品制备，以裂解目标病毒粒子/细胞和变性抑制剂，例如核糖核酸酶和蛋白酶有效，但也会抑制聚合酶活性，因此必须在使用前将其除去。 NAAT 工作流程的许多 POC 改编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：而不是自动化去除离液剂，我们将消除对其的需要，我们建议设计一种聚合酶。 “混沌”，或在离液放大缓冲液中活跃，能够同时提取，为了实现这一愿景，我们提出了三个方案。特定目标，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 靶点。目标 1：通过离液区室自我复制开发混沌聚合酶我们将使用高通量定向进化方法来开发保留保留的聚合酶。 3M GuSCN 中的活性（RNA 提取缓冲液的最低推荐浓度）。目标 2：通过深度突变扫描和研究研究聚合酶离液剂耐药机制我们将使用深度突变扫描和分子动力学模拟。研究 GuSCN 对聚合酶的相互作用并确定 GuSCN 抗性的合理设计方法。目标 3：将混沌聚合酶纳入执行样本的概念验证 HIV 诊断中使用性能最佳的单管进行裂解、RT-LAMP 扩增和比色检测。目标 1 和 2 中开发的混沌聚合酶，我们将设计一种用于 HIV 的单管 RT-LAMP 检测，以及针对纯 HIV RNA 和 HIV 患者血浆样本进行测试。