Live-cell imaging of small nucleolar RNA tagged with the broccoli aptamer in yeast

The development of the RNA 'vegetable' aptamers, Spinach and Broccoli, has simplified RNA imaging, especially in live cells. These RNA aptamers interact with a fluorophore (DFHBI or DFHBI-1T) to produce a green fluorescence signal. Although used in mammalian and Escherichia coli cells, the use of these aptamers in yeast has been limited. Here we describe how the Saccharomyces cerevisiae snoRNA, snR30, was tagged with the Spinach or the Broccoli aptamers and observed in live cells. The ability to observe aptamer fluorescence in polyacrylamide gels stained with a fluorophore or with a microplate reader can ease preliminary screening of the aptamers in different RNA scaffolds. In snR30 a tandem repeat of the Broccoli aptamer produced the best signal in vitro. Multiple factors in cell preparation were vital for obtaining a good fluorescence signal. These factors included the clearance of the native unmodified snR30, the amount and length of dye incubation and the rinsing of cells. In cells, the aptamers did not interfere with the structure or essential function of snR30, as the tagged RNA localized to the nucleolus and directed processing of ribosomal RNA in yeast. High-resolution images of the tagged snoRNA were obtained with live cells immobilized by a microcompressor.