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（NAATS）是用于传染病诊断的强大工具。而naats是它们在诊所通常使用，它们在护理点（POC）上下文中的使用受到复杂程序的约束需要从患者样品中提取和纯化核酸。这对于RNA特别麻烦靶标，由于RNA的脆弱性和样品中RNA降解色带的抽象环境。然而，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，大约2000万艾滋病毒居住）或快速周转和患者的自我测试很有用（例如，空中病毒爆发，例如影响力和新冠病毒）。在POC-NEAT环境中，需要更简单的RNA样品制备方法。 NAAT样品制备植物的许多复杂性来自自相矛盾的需要，将交际添加到提取核酸，然后在扩增前去除交际。诸如鸟肾之变硫氰酸酯（GUSCN）是破坏生物大分子结构的化学变性剂。他们用于NAAT样品制备中，以裂解目标病毒/细胞和成熟抑制剂，例如色带蛋白酶和蛋白酶。混沌是有效的，但也抑制聚合酶活性，因此必须将其去除放大。 NAAT工作流程的许多POC适应都涉及机器人或微流体自动化相反的增加和拆除，但仍需要专门的设备和/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：而不是自动化拆除了混乱，我们将消除对它的需求。我们建议设计一种聚合酶 “ Chaostable”或在交际放大缓冲液中活跃，可以同时提取，放大，并在单个管中检测病毒RNA靶标。要实现这一愿景，我们提出了三个具体目标，使用先前由我们的实验室和HIV-1开发的起始聚合酶作为模型RNA靶标。 AIM 1：通过Chaotrocic中的分隔自我复制开发可连接的聚合酶状况。我们将使用高通量定向进化方法来开发保留的聚合酶 3M GUSCN的活性（RNA提取缓冲液的最低建议浓度）。 AIM 2：通过深突变扫描和分子动力学模拟。我们将使用深突变扫描和分子动力学模拟研究GUSCN在我们的聚合酶上的相互作用，并确定GUSCN耐药性的合理设计方法。 AIM 3：将可连锁聚合酶纳入执行样品的概念验证HIV诊断单管中的裂解，RT-lamp扩增和比色检测。使用表现最佳在Aim 1和2中开发了可混合的聚合酶，我们将设计一个单管RT-LAMP测定法，用于HIV，并将其设计测试它针对纯HIV RNA和HIV患者血浆样品。