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Fluorescent RNA Technology

RNA is an emerging, rapidly growing research field in biological science and medicine. In addition to the well known functions of mRNAs, rRNAs, tRNAs in the central dogma of molecule biology, recent studies reveal the identity and functions of vast noncoding RNAs (ncRNAs) that play an important role in diverse biological processes, which are reshaping the prior conceptions about RNA functions. In living cells, RNAs exhibit complex dynamics including expression, degradation, translocation, splicing and various chemical modifications.

Methodology to visualize RNA such as is fluorescent in situ hybridization (FISH), enzymatic covalent labeling require cell fixation and are not suitable for live cell imaging. RNAs with engineered motifs can be tethered with fusions of fluorescent protein and specific RNA binding proteins (RBPs) e.g. MCP, PCP, λN or Cas may be used to image RNA in live cells at the single molecule level. However, the unbound MCP-FP molecules diffuse throughout the cells and generates high background fluorescence. In addition, whether such a heavy load of tethered protein affects the localization, stability and behavior of RNAs remains to be determined.

Figure 1. Technology for in situ RNA fluorescent labeling and detection.


In the history, fluorescent proteins (FPs) of different colors had revolutionized research of life sciences, which are genetically encoded labels of proteins enabling background free tracing of proteins in live cells. RNAs of interest may also be genetically labeled similarly and straightforwardly with fluorophore-binding RNA aptamers. These aptamers, termed fluorescent RNAs (FRs), shall also enable easy, robust, background free imaging of diverse RNAs in living cells.


While simple and as promising as they appear, however, the utility of FRs current available is limited. Some of the dye ligands of current FRs show significant background fluorescence in live cells and/or do not readily diffuse across plasma membranes, or the FRs. Some FRs has limited stability and brightness in live cells, or function as multimer. Analogously to FPs, ideal FRs should be monomeric, stable, bright, and multicolored, which had been challenging to achieve.

 Figure 2. A plattelet of Peppers with colors from cyan to red.


Peppers, a series of monomeric, highly bright, and stable FRs with a broad range of emission maxima spanning from cyan to red, were obtained by unique design of dendritic cell permeable dye ligand and multiple rounds of optimization. These dyes showed good membrane permeability, low cytotoxicity, and little fluorescence in solution or live cells. Compared to currently available FRs, Peppers showed an order of magnitude enhanced cellular fluorescence intensity and fluorescence turn-on ratio, one or two orders of magnitude enhanced affinity, ~20 oC increased Tm, expanded pH tolerance, and a broad spectral range available for live cell studies. For the first time, Peppers allow simple and robust imaging of mRNA and other RNA species in live cells with minimal perturbation of the target RNA’s transcription, localization, and translation. Peppers may also be used in imaging of genomic loci through CRISPR display, real-time tracking of protein-RNA tethering, and super-resolution imaging of RNA by structured illumination microscopy. Due to its high signal to background ratio, Peppers can be used in quantitative studies of RNAs in live cells, using flow cytometry or microplate reading. These Pepper FRs provide ideal tools for live imaging of cellular RNAs.

Figure 3. Pepper for background free RNA imaging and quantification in live cells. (a) Imaging of different RNAs in live cells. (b) Comparision of Pepper labeling efficiency with fluorescent protein. (c) Super-resolution imaging of Pepper. (d) Quantification of RNA and its protein in live cells using flow cytometry.



  1. Huang, K. et al. Structure-based investigation of fluorogenic Pepper aptamer. Nature chemical biology 17, 1289–1295, doi:10.1038/s41589-021-00884-6 (2021).
  2.  Chen, X. et al. Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat. Biotechnol. 37, 1287–1293, doi:10.1038/s41587-019-0249-1 (2019).