A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat–containing RNA

Abstract

Fluorescent imaging of RNA in living cells is a technically challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with ‘RNA mimics of GFP’. These are short aptamer tags that exhibit fluorescence upon binding otherwise nonfluorescent fluorophores that resemble those found in GFP. We find that the brightest of these aptamers, Spinach, often exhibits reduced fluorescence after it is fused to RNAs of interest. We show that a combination of thermal instability and a propensity for misfolding account for the low fluorescence of various Spinach-RNA fusions. Using systematic mutagenesis, we identified nucleotides that account for the poor folding of Spinach, and generated Spinach2, which exhibits markedly improved thermal stability and folding in cells. Furthermore, we show that Spinach2 largely retains its fluorescence when fused to various RNAs. Using Spinach2, we detail the cellular dynamics of the CGG trinucleotide-repeat containing “toxic RNA” associated with Fragile-X tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.

Metazoan tRNA introns generate stable circular RNAs in vivo

“…The resultant vectors are called pTRIC-Y and pTRIC-L (Y for tyrosine; L for leucine). To determine if pTRIC vectors can express heterologous tricRNAs, we inserted the Spinach2 RNA aptamer sequence (Strack et al 2013), transfected the constructs into human cells, and carried out qRT-PCR analysis. Figure 3D shows that Spinach2 expression was readily detectable using divergent PCR primer pairs.…”

“…Construction of the vectors expressing circular RNA aptamers pTRIC-Y:Sp2 and pTRIC-L:Sp2, which encode the Spinach2 RNA aptamer (Strack et al 2013), were constructed by cloning Spinach2 from a vector using PCR amplification and appropriate primers with the restriction sites NotI and SacII on the 5 ′ and 3 ′ ends of Spinach2, respectively. The primer sequences are as follows: tric_Sp2NotIf: CTGTG CGGCC GCGAT GTAAC TGAAT GAAAT GGTGA AGGAC GGG.…”

Microfluidic Formation of Monodisperse Coacervate Organelles in Liposomes

“…As Figure 4 a,b shows, the complex coacervates composed of spermidine and polyU together with IVTx components (see the Supporting Information, IVTx in artificial nucleoids, for details) were encapsulated into liposomes to prepare artificial cells with a nucleoid‐like structure. To visualize the RNA generation, we coded the DNA templates with a sequence for Spinach2 aptamer that can bind 5‐difluoro‐4‐hydroxybenzylidene imidazolinone (DFHBI) to form a fluorescent complex of Spinach2–DFHBI (Figure 4 a, last image);19 both DFHBI and Spinach2 are non‐fluorescent until binding occurs. The sequence of confocal images in Figure 4 c–e shows high fluorescence in the coacervates due to RNA synthesis, while a lower signal was observed in the surrounding water shells.…”

Computational analysis of fitness landscapes and evolutionary networks from in vitro evolution experiments

“…There are many examples of functional nucleic acid molecules that result from in vitro selections, which have been reviewed elsewhere [4]. For example, the Spinach2 aptamer [5] regulates the fluorescence of a fluorophore via binding, providing an interesting alternative to GFP tagging. In vitro selection has been applied to discover a variety of ribozymes [6], deoxyribozymes [7], aptazymes [8], and riboswitches [9].…”