Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters

Abstract

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

“…With their abilities to faithfully fold and function inside cells, aptamers can uniquely fill this niche for intracellular quantitative biology. Aptamers have been applied as reporters of intracellular metabolite levels [7; 8] and to image transcription and RNA localization in real time and in living cells [3; 9; 10]. …”

“…Although the MGA and SPN2A have similar affinities for their ligands, malachite green (MG) and many of its chemical variants are toxic to mammalian cells and yeast [13], DFHBI, the Spinach ligand is not toxic [3]. However, the in vivo signals received from Spinach [3; 9] and even the improved Spinach2 [10] are quite weak and not adequate for reporting on cellular mRNA levels [9]. The possibility of expanding the repertoire of aptamer reporters to enable the simultaneous tracking of more than one mRNA in a single cell motivated us to explore the use of light-up aptamers as a complement to IMAGEtags, from which a FRET signal is derived [9].…”

“…The second is the impact of tandem aptamers on fluorescent output and aptamer affinity. The outcome of this work is 1) identification of a new higher affinity ligand for Spinach aptamers with higher fluorescent yield than DFHBI, 2) demonstration that, when applied to imaging cells, several light-up aptamers give a high background fluorescence that can be largely overcome by using FRET-based aptamer reporters such as IMAGEtags [9], and 3) the finding that Spinach aptamers and MGAs perform independently when present as multiple tandem copies in a single RNA molecule and they do not cooperate synergistically for higher affinity or unit fluorescent yield.…”

Labelling and imaging of single endogenous messenger RNA particlesin vivo

“…Other recent approaches employ molecular beacon-like quenching schemes (Sunbul and Jäschke, 2013) or Förster resonance energy transfer (FRET)-based assays (Shin et al, 2014) to suppress fluorescence of unbound probes.…”

Hybrid Aptamer-Antibody Linked Fluorescence Resonance Energy Transfer Based Detection of Trinitrotoluene

“…The similar observations were also recorded in confocal micrographs after photobleaching experiments as shown in the insets of Figure 4c. The FRET efficiency calculated from photobleaching experiments was found to be ∼82% as calculated by the equation (FRET efficiency, E = 1 − F D /F D ′), 23 where F D and F D ′ are the donor intensities before and after photobleaching the acceptor, respectively. The newly developed sensing platform using the FITC-labeled aptamer as a binder molecule while anti-TNT antibody labeled with RITC as recognition element showed excellent sensitivity ∼0.4 nM for TNT detection.…”