Fiche publication
Date publication
août 2021
Journal
Nucleic acids research
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MELY Yves
,
Dr RICHERT Ludovic
,
Mr HUMBERT Nicolas
,
Pr DIDIER Pascal
Tous les auteurs :
Grytsyk N, Cianfarani D, Crégut O, Richert L, Boudier C, Humbert N, Didier P, Mély Y, Léonard J
Lien Pubmed
Résumé
Interconversions between nucleic acid structures play an important role in transcriptional and translational regulation and also in repair and recombination. These interconversions are frequently promoted by nucleic acid chaperone proteins. To monitor their kinetics, Förster resonance energy transfer (FRET) is widely exploited using ensemble fluorescence intensity measurements in pre-steady-state stopped-flow experiments. Such experiments only provide a weighted average of the emission of all species in solution and consume large quantities of materials. Herein, we lift these limitations by combining time-resolved fluorescence (TRF) with droplet microfluidics (DmF). We validate the innovative TRF-DmF approach by investigating the well characterized annealing of the HIV-1 (+)/(-) Primer Binding Sequences (PBS) promoted by a HIV-1 nucleocapsid peptide. Upon rapid mixing of the FRET-labelled (-)PBS with its complementary (+)PBS sequence inside microdroplets, the TRF-DmF set-up enables resolving the time evolution of sub-populations of reacting species and reveals an early intermediate with a ∼50 ps donor fluorescence lifetime never identified so far. TRF-DmF also favorably compares with single molecule experiments, as it offers an accurate control of concentrations with no upper limit, no need to graft one partner on a surface and no photobleaching issues.
Référence
Nucleic Acids Res. 2021 Aug 27;:
