Fluorophore‐Promoted RNA Folding and Photostability Enables Imaging of Single Broccoli‐Tagged mRNAs in Live Mammalian Cells

Abstract

Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence. Spinach/Broccoli‐DFHBI complexes exhibit high fluorescence in vitro, but they exhibit lower fluorescence in mammalian cells. Here, computational screening was used to identify BI, a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands. BI prevents thermal unfolding of Broccoli at 37 °C, leading to more folded Broccoli and thus more fluorescent Broccoli‐BI complexes in cells. Broccoli‐BI complexes are more photostable owing to impaired photoisomerization and rapid unbinding of photoisomerized cis‐BI. These properties enable single mRNA containing 24 Broccoli aptamers to be imaged in live mammalian cells treated with BI. Small molecule ligands can thus promote RNA folding in cells, and thus allow single mRNA imaging with fluorogenic aptamers.

Concatemeric Broccoli reduces mRNA stability and induces aggregates

“…This question has been addressed by Li et al . and they determined no effect of the repeated Broccoli RNA aptamer on mRNA stability [ 10 ]; quite different to our experience with it.…”

“…The most successful approach so far in this context though was carried out by Li et al . utilizing a newly designed substrate, termed BI, that increases Broccoli folding and stability [ 10 ]. Future studies are directed to evaluate more advanced substrates in our experimental setting.…”

“…Single aptamer tags so far have not been successful in the tracking or quantification of single molecules though, but reminiscent of the MS2-tag concatemers, tandem arrays could greatly empower the scope of the methodology. Recently, the tandem array approach has been successfully utilized, in bacteria [ 9 ], and, with a new substrate, to visualize overexpressed RFP carrying mRNA molecules in mammalian cells [ 10 ]. However, the introduction of multiple extensively structured exogenous sequences into a given RNA molecule may alter the physiological behavior of the respective molecules.…”

Studying SARS-CoV-2 with Fluorescence Microscopy

“…It is based on RNA aptamers specifically binding cell-permeable fluorogenic dyes, which are non-fluorescent in solution but become brightly fluorescent (>1000-fold fluorescence increase) in a complex with the cognate aptamer [ 70 ]. While the early versions of aptamers (e.g., Spinach) suffered from low stability and high thermal and ion sensitivity, recent improvements of both aptamers (e.g., Broccoli) and fluorogens resulted in robust labeling of target RNAs in mammalian cells [ 71 ]. A possibility of imaging single RNA molecules inside the Cos-7 cells was demonstrated with the fluorogenic Mango II arrays [ 72 ].…”

Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase‐Modified Fluorogenic RNA Aptamers

“…The apta‐FRET concept is based on assembling two fluorogenic aptamers, Spinach and Mango, on an RNA origami framework, enabling energy transfer from DFHBI‐1T bound to Spinach, to YO3‐biotin complexed by Mango. The knowledge about the structure of the aptamers not only facilitates the optimization of photophysical properties but is also essential for the design of functional devices that depend on the relative orientation of aptamers and their bound ligands …”