Spatiotemporally controlled generation of NTPs for single-molecule studies.
Nat Chem Biol
; 18(10): 1144-1151, 2022 10.
Article
em En
| MEDLINE
| ID: mdl-36131148
ABSTRACT
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Nucleossomos
/
Nucleosídeos
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article