RNA-based fluorescent biosensors for live cell imaging of small molecules and RNAs

Abstract

Genetically encodable fluorescent biosensors provide spatiotemporal information on their target analytes in a label-free manner, which has enabled the study of cell biology and signaling in living cells. Over the past three decades, fueled by the development of a wide palette of fluorescent proteins, protein-based fluorescent biosensors against a broad array of targets have been developed. Recently, with the development of fluorogenic RNA aptamer-dye pairs that function in live cells, RNA-based fluorescent (RBF) biosensors have emerged as a complementary class of biosensors. Here we review the current state-of-the-art for fluorogenic RNA aptamers and RBF biosensors for imaging small molecules and RNAs, and highlight some emerging opportunities.

Figure 1.

Fluorogenic RNA aptamers-dye pairs. Above, schematic representations of two types of fluorogenic RNA aptamers based on their paired dyes: single dyes (A) and fluorophore-quencher conjugates (B). Yellow star, fluorophore; Gray rounded rectangle, quencher. Below, chemical structures of representative dyes. DFHBI, 3,5-difluoro-4-hydroxybenzylidene imidazolinone [8]; DFHBI-1T, DFHBI with a 1,1,1-trifluoroethyl substituent [9]; DFHO, 3,5-difluoro-4-hydroxybenzylidene imidazolinone-2-oxime [15]; TO, thiazole orange [18]; YO, oxazole yellow [20]; DIR, dimethylindole red [7]; OTB, oxazole thiazole blue [7]; SiR, silicon rhodamine [21]; HBC, (4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenyl-acetonitrile [22]; SR, Sulforhodamine B; DN, dinitroaniline; TMR, 5-carboxytetramethylrhodamine; MN, mononitroaniline [25,26]; BHQ, black hole quencher; Cbl, cobalamin [23].

Figure 2.

Design strategies of RNA-based fluorescent biosensors for molecular sensing. Functional RBF biosensors can be generated either by (A) splitting the dye-binding aptamer [16] or (B) splitting the ligand-sensing aptamer [•]. (C) Fluorogenic riboswitches can be generated by replacing the regulatory expression platform in natural riboswitches with a dye-binding aptamer [34]. (D) Ligand-sensing aptamer can be inserted into an established RNA origami scaffold for ligand-dependent FRET signal change [•]. (E) Allosteric ribozymes can be fused to a dye-binding aptamer for ligand-dependent release of the fluorogenic aptamer [36]. Red circle, target ligand; dashed square, fusion section between the ligand-sensing domain and the signal reporter domain. Brown triangle, self-cleavage site of ribozyme.

Figure 3.

Examples of RBF biosensors for RNA sensing. (A) Split-binding domain strategy was applied on Spinach and SRB-2 aptamer to generate biosensors for miRNA [•,•]. (B) Destabilizing the fluorogenic aptamer by shortening a stem or splitting led to functional biosensors for mRNA [28,53]. (C) For sensitive RNA detection, the catalytic hairpin assembly was used to amplify the signal triggered by target RNA [55].