Cell lines

293T/17 cells

Preparation Method

293T/17 cells were transfected with the corresponding plasmids. After 30h, the cells were digested and resuspended in the 4% FBS/1× PBS solution containing 0.2μM HBC and 5mM MgSO₄. The fluorescence was analyzed using a CytoFLEX-S flow cytometer.

Reaction Conditions

0.2μM HBC

Applications

When cells co-expressing Pepper-tagged centromere-targeting sgRNA and dCas9-GFP were incubated with the HBC620 fluorophore, numerous (~40) nuclear foci of Pepper620 fluorescence nearly perfectly co-localized with GFP fluorescence.

HBC620 is an analog of HBC, which is a GFP fluorophore-like synthetic dye with a structurally rigid electron acceptor and a strong electron donor. HBC is nonfluorescent in solution, but strongly fluoresces upon constraining intramolecular motion. HBC-Pepper complex can be used to visualize RNA dynamics in live cells^[1].

HBC's fluorescence can be enhanced more than 3000-fold in vitro by Pepper. Mammalian cells expressing Pepper exhibited robust greenyellowish fluorescence on incubation with 1μM HBC^[1].

References:
[1].Chen X, Zhang D, et al. Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat Biotechnol. 2019;37(11):1287-1293.