Bright, fluorogenic and photostable avidity probes for RNA imaging

Abstract

Fluorescent light-up aptamers (FLAPs) emerged as valuable tools to visualize RNA, but are mostly limited by poor brightness, low photostability and high fluorescence background. In this study, we combine bivalent (silicon) rhodamine fluorophores with dimeric FLAPs to yield bright and photostable complexes with low picomolar dissociation constants. Our avidity-based approach resulted in extreme binding strength and high fluorogenicity, enabling single mRNA tracking in living cells.

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

“…TMR 2 contains bivalent TMR dyes, which are essentially non‐fluorescent due to intramolecular H‐dimer formation (Figure 4, 5 and Table 1, 2). In contrast, biRhoBAST can bind TMR 2 and disrupt intramolecular H‐dimers, resulting in a 55‐fold increase in fluorescence [24] …”

“…However, the cellular background of the TMR 2 is high even though the fluorophore concentration is as low as 500 nM. In all, the biRhoBAST: TMR 2 system is the brightest fluorescent light‐up aptamers reported to date [24] …”

“…These phenomena suggest that TMR 2 is capable of imaging biRhoBAST‐tagged mRNAs in living mammalian cells (Figure 2e). [24] …”

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

“…In contrast, biRhoBAST can bind TMR 2 and disrupt intramolecular H-dimers, resulting in a 55-fold increase in fluorescence. [24] Compared to monomeric RhoBAST: TMR-DN system, biRhoBAST: TMR 2 system has a strong affinity (K D = 0.04 nM), high photostability with only a 3 % decrease after 90s illumination, slow dissociation (k off = 3.8 × 10 À 4 s À 1 ) with a � 10 À 4 fold slower than the monomeric RhoBAST: TMR-DN system (k off = 2.9 s À 1 ), high fluorescence (ϕ = 0.88, ɛ = 165 000 M À 1 cm À 1 ) (Table 1), and low magnesium concentra-tion-dependent affection. However, the cellular background of the TMR 2 is high even though the fluorophore concentration is as low as 500 nM.…”

Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging

“…Popular FLAP systems such as Spinach 6 , Broccoli 7 and Pepper 8 are based on dyes that are essentially non-fluorescent in solution but acquire fluorescence upon aptamer binding due to effective suppression of their intramolecular dynamics. Furthermore, FLAPs such as RhoBAST, SiRA, DNB and Riboglow have been developed that are based on photostable, bright and intrinsically fluorescent dyes, notably rhodamines [9][10][11][12][13][14][15][16] . High brightness and low apparent photobleaching of RhoBAST-and SiRA-based aptamer:dye complexes have allowed their use as genetically encoded probes for super-resolution imaging, including singlemolecule localization microscopy (SMLM) and stimulated emission depletion (STED) microscopy 12,13 .…”