High Accuracy Nanopore Sequencing

高精度纳米孔测序

• 批准号: 10267319
• 负责人: JENS GUNDLACH
• 金额: $ 31.36万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10267319
• 关键词: 2019-nCoV; Antiviral Agents; Award; Biological Assay; COVID-19; DNA; DNA Sequence; DNA sequencing; Drug Design; Drug Targeting; Enzyme Inhibitor Drugs; Enzyme Kinetics; Enzymes; Exonuclease; Funding; Genome; Genomics; Grant; Kinetics; Knowledge; Measures; Methods; Monitor; Motion; Motor; National Human Genome Research Institute; Nonstructural Protein; Nucleic Acids; Nucleotides; Pharmaceutical Preparations; Physiological; Polymerase; Positioning Attribute; Proteins; RNA; RNA-Directed RNA Polymerase; Research; Resolution; Site; Techniques; Technology; Time; Viral; Viral Genome; Virus; Virus Replication; design; drug candidate; helicase; improved; in vivo; inhibitor/antagonist; insight; millisecond; nanopore; pandemic disease; premature; prevent; remdesivir; single molecule; stem; tool; viral RNA

This project is an expansion of the scope of our current funding which was awarded to improve nanopore sequencing. In the course of our research to improve nanopore DNA sequencing, we have discovered and developed a new high-resolution single-molecule tool to observe the motion of helicases and polymerases. The tool, which we called Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), is able to reveal the single-nucleotide steps of helicases and polymerases at unprecedented detail. These enzyme steps are so small (~0.3 nm) and fast (~ 1 ms) that no other existing single-molecule technique can resolve such motion in real time and at physiological conditions. The genome of the SARS-CoV-2 virus encodes for an RNA-dependent RNA polymerase, also known as non-structural protein 12 (nsp12), and a superfamily 1B helicase, known as nonstructural protein 13 (nsp13). Both of these enzymes are essential and specific to the in vivo replication of many viruses, making this genomic replication machinery an ideal drug target. Several drug candidates to inhibit these enzymes exist, however, none are as effective as they need to be to stem the tide of the current pandemic. Many questions remain about how exactly these drugs interfere with the function of nsp12 or nsp13. SPRNT provides an opportunity to answer these questions. We aim to use SPRNT to analyze the single-molecule motion of nsp12 and nsp13 in physiological conditions and in the presence of drugs against them. The exact knowledge of the mechanisms by which various drugs inhibit nsp12 or nsp13 will enable more rational and rapid design of effective antiviral drugs that have the potential to stop COVID-19.

该项目是我们当前资助范围的扩展，该资助旨在改善纳米孔 测序。在我们改进纳米孔 DNA 测序的研究过程中，我们发现并 开发了一种新的高分辨率单分子工具来观察解旋酶和聚合酶的运动。这 我们称之为单分子皮米分辨率纳米孔镊子 (SPRNT) 的工具能够揭示 解旋酶和聚合酶的单核苷酸步骤的细节前所未有。这些酶的步骤是如此之小 （~0.3 nm）和快速（~ 1 ms），没有其他现有的单分子技术可以实时解决这种运动 以及在生理条件下。 SARS-CoV-2 病毒的基因组编码依赖于 RNA 的 RNA 聚合酶，也称为非结构蛋白 12 (nsp12)，以及超家族 1B 解旋酶，称为非结构蛋白 13 (nsp13)。这两种酶对于许多细胞的体内复制都是必需的和特异的。 病毒，使这种基因组复制机制成为理想的药物靶点。几种抑制这些的候选药物 然而，酶是存在的，但没有一种酶能够像阻止当前大流行的浪潮那样有效。许多 这些药物究竟如何干扰 nsp12 或 nsp13 的功能仍存在疑问。 SPRNT 提供 有机会回答这些问题。我们的目标是利用SPRNT来分析nsp12的单分子运动 和 nsp13 在生理条件下和存在针对它们的药物的情况下。准确的知识 各种药物抑制 nsp12 或 nsp13 的机制将有助于更合理、更快速地设计有效的药物 有可能阻止 COVID-19 的抗病毒药物。