Inert Pepper aptamer-mediated endogenous mRNA recognition and imaging in living cells

Abstract

The development of RNA aptamers/fluorophores system is highly desirable for understanding the dynamic molecular biology of RNAs in vivo. Peppers-based imaging systems have been reported and applied for mRNA imaging in living cells. However, the need to insert corresponding RNA aptamer sequences into target RNAs and relatively low fluorescence signal limit its application in endogenous mRNA imaging. Herein, we remolded the original Pepper aptamer and developed a tandem array of inert Pepper (iPepper) fluorescence turn-on system. iPepper allows for efficient and selective imaging of diverse endogenous mRNA species in live cells with minimal agitation of the target mRNAs. We believe iPepper would significantly expand the applications of the aptamer/fluorophore system in endogenous mRNA imaging, and it has the potential to become a powerful tool for real-time studies in living cells and biological processing.

Single mRNA Imaging with Fluorogenic RNA Aptamers and Small‐molecule Fluorophores

“…These small molecule‐binding RNA sequences mostly do not exist in endogenous mRNA, and usually are fused in 3′UTR or 5′UTR. However, using the split aptamer [25] or conformation‐switching induced RNA aptamers [26] approach, endogenous mRNA imaging could be developed with RNA aptamers: SFs systems. Fourth, several small molecule‐binding RNA sequences may have artificial structures, such as RNA G‐quadruplex (RG4), resulting in the recruitment of the helicase protein and affecting the destiny of target mRNA [27]…”

Single mRNA Imaging with Fluorogenic RNA Aptamers and Small‐molecule Fluorophores

“…[41] For example, the imaging systems were used for non-coding RNA and mRNA aggregates/granules imaging. [26] In addition, the imaging systems were constructed as various RNA-based sensors for small-molecule metabolite, protein, ions detection in living cells. [42] Moreover, the systems can be engineered for chromosomal imaging with CRISPR.…”

Insights of Life Molecules’ Dynamic Distribution in Live Cells via Sequence–Structure Bispecific Fluorescent RNA

“…Fluorescent RNAs (FLRNAs), − the complex of RNA aptamers with a cognate dye ligand, enable us to activate the fluorescent dye in which the RNA aptamer could be labeled onto proteins directly, connected with nucleic acids or custom-designed with various aptamers to image biomolecules in cells. − However, most of FLRNAs still suffer from low brightness, high background, mainly G-tetrad RNA modules, and limited availability of aptamer–dye combinations. The low specificity, presence of a large number of intracellular G-tetrad sequences, and scarcity of aptamer–dye combinations seriously hamper the application of RNA G-tetrad modules in vivo .…”