Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution

Abstract

Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse applications, including imaging RNA trafficking and as a component of RNA-based sensors that exhibit fluorescence upon binding small molecules in live cells. These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the fluorescence of fluorophores similar to that found in green fluorescent protein. Although additional highly fluorescent RNA–fluorophore complexes would extend the utility of this technology, the identification of novel RNA–fluorophore complexes is difficult. Current approaches select aptamers on the basis of their ability to bind fluorophores, even though fluorophore binding alone is not sufficient to activate fluorescence. Additionally, aptamers require extensive mutagenesis to efficiently fold and exhibit fluorescence in living cells. Here we describe a platform for rapid generation of highly fluorescent RNA–fluorophore complexes that are optimized for function in cells. This procedure involves selection of aptamers on the basis of their binding to fluorophores, coupled with fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in Escherichia coli. Promising aptamers are then further optimized using a FACS-based directed evolution approach. Using this approach, we identified several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one. Broccoli shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2. This reflects, in part, improved folding in the presence of low cytosolic magnesium concentrations. Thus, this novel fluorescence-based selection approach simplifies the generation of aptamers that are optimized for expression and performance in living cells.

Metazoan tRNA introns generate stable circular RNAs in vivo

“…3A). For these experiments, we used a novel in-gel imaging procedure to monitor transcription and intron circularization of another GFP-mimicking RNA aptamer, called Broccoli (Filonov et al 2014). Samples were run on a denaturing polyacrylamide gel, renatured in the gel, and stained with DFHBI-1T to visualize Broccoli (Fig.…”

“…RiboRuler Low Range RNA Ladder (Thermo Scientific) was used to calibrate molecular weight. The position of Broccoli-containing bands can be visualized by staining the gel with DFHBI-1T, which specifically binds to Broccoli only, using a previously published method (Filonov et al 2014). Briefly, after electrophoresis gels were washed 3 × 5 min with water and then stained for 30 min in 10 µM DFHBI-1T in a buffer containing 40 mM HEPES pH 7.4, 100 mM KCl, 1 mM MgCl 2 .…”

The emerging molecular biology toolbox for the study of long noncoding RNA biology

“…Even though there are numerous strategies to optimize smFISH for lncRNA detection, there are occasions where the lncRNA may not be amenable to detection by this method. In these extreme cases, it may be worth investigating the use of gene editing technologies to tag lncRNA transcripts with RNA mimics of GFP [41,42]. However, the consequences of adding such structured sequences to lncRNAs are unknown and may very well affect the function of the transcript.…”

Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein Using Fluorescence-Activated Cell Sorting

“…It has been reported that Spinach/DFHBI displays fluorescence properties similar to GFP (*50 % brightness of EGFP), and therefore, this system represents a useful tool to examine RNA activities in cells (Paige et al 2012(Paige et al , 2011Song et al 2014;. The same group has also engineered another aptamer, named ''Broccoli,'' that can also bind and activate DFHBI (Filonov et al 2014). Broccoli was selected using a combined approach of conventional SELEX and fluorescence-activated cell sorting (FACS).…”