4.8 Article

Spatiotemporally controlled generation of NTPs for single-molecule studies

Journal

NATURE CHEMICAL BIOLOGY
Volume 18, Issue 10, Pages 1144-+

Publisher

NATURE PORTFOLIO
DOI: 10.1038/s41589-022-01100-9

Keywords

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Funding

  1. European Research Council (ERC) Starting Grant [ERC-StG-714068]
  2. Knut and Alice Wallenberg Foundation [KAW/WAF 2019.0306, KAW 2016.0077]
  3. Cancerfonden [19 0055 Pj]
  4. Swedish Research Council [VR 03534]
  5. Ministry of Science and Higher Education of the Russian Federation [075-15-2021-1333]

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The study introduces a method called LAGOON for controlling NTP-driven reactions in single-molecule experiments by locally generating NTPs. This method significantly increases measurement throughput and enables single-turnover observations. The effectiveness of LAGOON is demonstrated in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding, and RNA polymerase elongation. LAGOON can be easily integrated with various single-molecule techniques, and is expected to facilitate studies on a wide range of crucial NTP-driven processes.
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.

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