BI is a DFHBI (3, 5-difluoro-4-hydroxybenzylidene imidazolinone) derivative that binds Broccoli™ with higher affinity and deliver improved cellular fluorescence. In cells, BI has been found to stabilize the Broccoli™ structure and promote its folding. Additionally, Broccoli™/BI complex is significantly more photostable due to impaired light-induced photoisomerization, and rapid unbinding of photoisomerized cis-BI. BI is cell-permeable with negligible toxicity in living cells and can be used to label any genetically encoded Broccoli™ RNA tag without disrupting biological functions. Importantly, the optimized fluorescence properties of BI enable live single-molecule imaging of Broccoli™-tagged mRNA transcripts in mammalian cells. Compared to MS2-tagged mRNA, Broccoli™-tagged mRNA puncta exhibit similar size and puncta diameters fall within a single Gaussian curve./

Data/



Figure 1. Live-cell imaging of COS7 cells expressing β-actin mRNA transcripts tagged with 24 copies of Broccoli™ in the 3’UTR. Cells were incubated with 0.5 µg/ml Hoechst 33342 stain (blue) and 10 µM BI (green) and images were acquired using a 500 msec exposure with a GFP filter set. Mobile fluorescence puncta were observed only when Broccoli-expressing cells were treated with BI./



Figure 2. For single-molecule imaging, COS7 cells were transfected with a construct expressing mCherry-24xBroccoli™ transcripts and imaged in the presence 10 µM BI. Images of the same cells were acquired using TRITC and GFP filter sets to capture mCherry fluorescence (red) and Broccoli™ (green) signals. Mobile fluorescent puncta were detected in both the nuclei and cytosol of mCherry-expressing cells./



Figure 3. Live fluorescent mRNA puncta were imaged using COS7 cells expressing either A. the MS2-GFP system and B. the Broccoli™-BI system. The puncta size of Broccoil-tagged mRNA is similar to the puncta size of MS-tagged mRNA. Further, the puncta intensities of mRNA labeled from both systems exhibit a single Guassian distribution, consistent with the majority of these puncta reflecting single mRNA transcripts. The scale bars, are both 5 µm.