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Sabantsev A, Mao G, Aguirre Rivera J, Panfilov M, Arseniev A, Ho O, Khodorkovskiy M, Deindl S
Nat. Chem. Biol. 18 (10) 1144-1151 [2022-10-00; online 2022-09-21]
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.
PubMed 36131148
DOI 10.1038/s41589-022-01100-9
Crossref 10.1038/s41589-022-01100-9
pii: 10.1038/s41589-022-01100-9
pmc: PMC9512701