DFHBI

DFHBI is a small molecule that resembles the chromophore of green fluorescent protein (GFP). DFHBI can be used for imaging RNA in living cells. . Spinach and DFHBI are essentially nonfluorescent when unbound, whereas the Spinach-DFHBI complex is brightly fluorescent both in vitro and in living cells (Ex/Em = 469/501 nm). In Vitro:1. Solution preparation^[3]
1.1 Preparation of stock solution
Solvent: DMSO
Concentration: 20 mM (optimized according to the experiment).
Storage: Store at -20°C or -80°C in dark after aliquoting. Avoid repeated freezing and thawing.
1.2 Preparation of working solution
Dilute to 20 µM with PBS or cell culture medium (pH 7.4) (optimized according to the experiment).
Note: The working solution should be prepared and used immediately. Keep it away from light.

2. Cell staining
2.1 HEK293T cells are transfected with pAV-5S.
2.2 Add 20 µM DFHB into the cell plate.
2.3 Incubate the dye-loading plate in a cell incubator for 30 minutes.
2.4 Fluorescence microscopy detection (Ex/Em = 469/501 nm).

These RNAs interact with DFHBI to produce a bluish-green fluorescence emission (501 nm) after excitation at 447 nm. Spinach and Spinach2 are RNA aptamers that can be used for the genetic encoding of fluorescent RNA. Spinach2 binds and activates the fluorescence of DFHBI, allowing the dynamic localizations of Spinach2-tagged RNAs to be imaged in live cells. The spectral properties of Spinach2 are limited by DFHBI, which produces fluorescence that is bluish-green and is not optimized for filters commonly used in fluorescence microscopes. Spinach and Spinach2 bind to DFHBI have fluorescence excitation maxima of 447 nm and peak fluorescence emission of 501 nm^[1].