Abstract
Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer–fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8–64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.
Highlights
The second type of RNA imaging utilizes indirect labeling and employs fluorescent fusion proteins and specific protein-RNA interactions, such as the RNA bacteriophage MS2 coat protein system[16,17,18], the PP7 bacteriophage system[19,20], the bacteriophage λ N coat protein system[21], etc.[22,23]
The Spinach system is a recently developed RNA labeling and imaging method based on aptamer binding and fluorescence induction of the fluorogenic small molecule DFHBI34
There were few reports on single Spinach aptamer labeling for cellular mRNA imaging[42], likely due to its low fluorescence brightness suggested by our experimental data (Fig. 2D)
Summary
The second type of RNA imaging utilizes indirect labeling and employs fluorescent fusion proteins and specific protein-RNA interactions, such as the RNA bacteriophage MS2 coat protein system[16,17,18], the PP7 bacteriophage system[19,20], the bacteriophage λ N coat protein system[21], etc.[22,23]. In both in vitro measurement and cellular imaging, the Spinach array brought about as high as 17-fold fluorescence enhancement compared to single Spinach aptamer on RNAs, allowing us to image mRNA distributions inside living cells that could have not been achieved with single aptamer tagging.
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