Co-evolution of genetically encoded light-up aptamers and fluorogenic probes for live-cell super-resolution RNA imaging

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Imaging RNA in intact cells reveals the processes of transcription, translation, gene expression and regulation with high temporal and spatial resolution. Unfortunately, the imaging and detection of RNA in vivo has been hampered by the lack of intrinsically fluorescent RNAs. The currently available light-up aptamers still lack the sensitivity to image single RNA molecules with high spatial and temporal resolution in vivo. Our goal is to develop super-resolution RNA imaging techniques with single-molecule sensitivity that allow studying RNA biochemistry in vivo. Based on the progress made in the first funding period, we aim to:i) evolve SiRA and SRB-4 aptamers using a combination of SELEX and FACS techniques to improve the fluorescence turn-on factors as well as dissociation constants.ii) synthesize next-generation rhodamine-based fluorophores with high quantum yields and phostability based on Janelia dyes for SiRA, SRB-4, and their variants. iii) image distinct RNA molecules in live cells using SiRA and SRB-4 light-up aptamers combined with new probes using super-resolution microscopy (STED and STORM).iv) utilize the FRET concept to improve the sensitivity for RNA detection in fluorescence microscopy. (SRB-4 and SiRA are not only orthogonal to each other, but also a good FRET pair). v) explore different concepts (namely rotational quenching and color-shifting fluorophores) to generate new classes of light-up aptamers. vi) to develop super-dark probes which do not fluorescence unless they bind to the aptamer target.vii) utilize the “avidity” concept for single-molecule RNA imaging, where new RNA scaffolds will be employed to pre-organize aptamer dimers for probe binding.