High Accuracy Nanopore Sequencing

这个项目是我们目前的资助范围的扩大，该资助被授予改善纳米孔 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。的 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，在前所未有的细节。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在真实的时间内解决这种运动 和生理条件下。SARS-CoV-2病毒的基因组编码依赖RNA的RNA 聚合酶，也称为非结构蛋白12（nsp 12），和超家族1B解旋酶，称为非结构蛋白13（nsp 13）。这两种酶都是必需的，并且对许多人的体内复制具有特异性。 病毒，使这种基因组复制机制成为理想的药物靶点。几种候选药物抑制这些 酶是存在的，但是，没有一种酶能像它们所需要的那样有效地阻止当前的流行病。许多 关于这些药物究竟如何干扰nsp 12或nsp 13的功能，仍然存在问题。SPRNT提供 回答这些问题的机会我们的目的是利用SPRNT来分析nsp 12的单分子运动 和nsp 13的活性。对这一事件的确切了解 各种药物抑制nsp 12或nsp 13的机制将使得能够更合理和快速地设计有效的 有可能阻止COVID-19的抗病毒药物。

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.