Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes.

细胞中的许多基本过程都依赖于使用三磷酸核苷 (NTPs) 的蛋白质。在单分子水平上直接监测这些蛋白质通常复杂的动力学的方法有助于揭示它们的作用机制。然而，对于利用 NTP 的反应，测量吞吐量通常受到限制，并且对多个连续周转的复杂动力学进行定量剖析仍然具有挑战性。在这里，我们提出了一种通过本地生成 NTP (LAGOON) 在单分子实验中控制 NTP 驱动反应的方法，该方法显着提高了测量吞吐量并实现了单周转观察。我们通过监测 DNA 解旋、核小体滑动和 RNA 聚合酶延伸来证明 LAGOON 在单分子荧光和力光谱分析中的有效性。