Department of Physics, University of Washington, Seattle, WA 98195, U.S.A.
Essays Biochem. 2021 Apr 16;65(1):109-127. doi: 10.1042/EBC20200027.
Single-molecule picometer resolution nanopore tweezers (SPRNT) is a technique for monitoring the motion of individual enzymes along a nucleic acid template at unprecedented spatiotemporal resolution. We review the development of SPRNT and the application of single-molecule kinetics theory to SPRNT data to develop a detailed model of helicase motion along a single-stranded DNA substrate. In this review, we present three examples of questions SPRNT can answer in the context of the Superfamily 2 helicase Hel308. With Hel308, SPRNT's spatiotemporal resolution enables resolution of two distinct enzymatic substates, one which is dependent upon ATP concentration and one which is ATP independent. By analyzing dwell-time distributions and helicase back-stepping, we show, in detail, how SPRNT can be used to determine the nature of these observed steps. We use dwell-time distributions to discern between three different possible models of helicase backstepping. We conclude by using SPRNT's ability to discern an enzyme's nucleotide-specific location along a DNA strand to understand the nature of sequence-specific enzyme kinetics and show that the sequence within the helicase itself affects both step dwell-time and backstepping probability while translocating on single-stranded DNA.
单分子皮米分辨率纳米孔镊子（SPRNT）是一种在前所未有的时空分辨率下监测单个酶沿着核酸模板运动的技术。我们回顾了 SPRNT 的发展以及将单分子动力学理论应用于 SPRNT 数据，以开发沿着单链 DNA 底物运动的解旋酶的详细模型。在本综述中，我们提出了 SPRNT 可以在超家族 2 解旋酶 Hel308 背景下回答的三个问题。对于 Hel308，SPRNT 的时空分辨率能够分辨出两种不同的酶亚基，一种依赖于 ATP 浓度，另一种则与 ATP 无关。通过分析停留时间分布和解旋酶回溯，我们详细展示了 SPRNT 如何用于确定观察到的这些步骤的性质。我们使用停留时间分布来区分解旋酶回溯的三种不同可能模型。最后，我们使用 SPRNT 区分酶在 DNA 链上核苷酸特异性位置的能力来了解序列特异性酶动力学的性质，并表明解旋酶本身的序列会影响单链 DNA 上的单步停留时间和回溯概率。
