3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime-1-benzoimidazole ( OBI )

OBI is a DFHBI (3, 5-difluoro-4-hydroxybenzylidene imidazolinone) derivative that binds either Broccoli™ or Red Broccoli™ and generates red fluorescence. Conversely, Red Broccoli™ also binds BI to emit green fluorescence. This “plug-and-play” capability allows the spectral properties of fluorogenic aptamers and fluorophores to be altered based on the specific experimental needs of the users. In the cells, OBI binds Red Broccoli™ with higher affinity and makes it resistant to thermal unfolding. This enables the Red Broccoli™/OBI complex to be readily detectable in live mammalian cells. This is particularly useful because green fluorescent probes suffer from the cellular green background fluorescence derived from endogenous vitamins and co-factors. Thus, red fluorescent RNAbased tags and probes would improve the accuracy of intracellular imaging.


Red Broccoli™-OBI complex exhibits increased photostability in cells and in vitro. In-cell photostability of Red Broccoli™-fluorophores (OBI versus BI, 20 µM each) was assessed by continuous illumination of Red Broccoli™-expressing cells. Red Broccoli™-OBI maintained >75% of its red fluorescence for over 10 s, while Red Broccoli™-BI lost ~50% of its green fluorescence within ~0.2 s. Scale bar, 10 µm.


The enhanced cellular stability of OBI enables imaging of endogenous SAM dynamics in mammalian cells using a Red Broccoli™-based SAM biosensor. (a) A schematic diagram of the SAM sensor used in this experiment. (b) HEK293T cells expressing either the Red Broccoli™-based SAM sensor (top) or Red Broccoli™ alone (bottom) were imaged with 10 µM OBI. Sensor-expressing cells treated with 50 mM cycloleucine at 0 min to block SAM biosynthesis exhibited rapidly decreases in cellular fluorescence. After 120 min, the media was replaced with cycloleucine-free media (“Wash out”) and fluorescence recovered to baseline levels over time. Cells expressing only Red Broccoli™ maintained consistent red fluorescence over time. Scale bar, 20 µm.


BI and OBI are “plug-and-play” fluorophores for Broccoli™ and Red Broccoli™. HEK293T cells expressing Red Broccoli, Broccoli, or control RNA were incubated with 10 µM OBI (a-c) or BI (d-f). OBI exhibited red fluorescence in both Red Broccoli- and Broccoli-expressing cells and BI exhibited green fluorescence in both Red Broccoli- and Broccoli-expressing cells. But no fluorescence was seen in control RNA-expressing cells with either fluorophore. Cell nucleus was labeled with Hoechst-33342 stain (blue) and imaged with DAPI and a FITC filter cube for BI or TRITC filter cube for OBI. Scale bar, 10 µm.

Please contact us at for specific academic pricing.

More Details