RhoBAST is a novel fluorescence light-up RNA aptamer (FLAP) that transiently binds a fluorogenic rhodamine dye. Fast dye association and dissociation result in intermittent fluorescence emission, facilitating single-molecule localization microscopy (SMLM) with an image resolution not limited by photobleaching. We demonstrate RhoBAST's excellent properties as a RNA marker by resolving subcellular and subnuclear structures of RNA in live and fixed cells by SMLM and structured illumination microscopy (SIM)..

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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.

The structural and functional properties of zinc(ii) complexes of two nitrogen rich polydentate ligands, HTPDP = 1,3-bis(bis-pyridin-2-ylmethylamino)propan-2-ol and HTPPNOL = N,N,N'-tris-(2-pyridylmethyl)-1,3-diaminopropan-2-ol, are compared. HTPDP is a hepta-dentate ligand with four pyridyl groups attached to a 1,3-diaminopropan-2-ol backbone while HTPPNOL contains only three pyridyl groups. In reactions with Zn(ClO), HTPDP forms a dinuclear zinc compound [Zn(TPDP)(OAc)](ClO), 1. On the other hand, mononuclear [Zn(HTPPNOL)](ClO), 2, and tetranuclear [Zn(TPPNOL)(OAc)](ClO), 3, complexes were isolated with the ligand HTPPNOL. Kinetic measurements with the substrate bis(2,4-dinitrophenyl)phosphate (BDNPP) revealed that compound 1 (k = 31.4 × 10 min) is more reactive than 3 (k = 7.7 × 10 min) at pH = 8.5, whilst the mononuclear compound 2 is inactive. Compound 1 displays a typical steady-state kinetic behaviour, while compound 3 exhibits steady-state behaviour only ∼120 s after starting the reaction, preceded by a burst-phase. P NMR studies confirm that 1 can promote the hydrolysis of both ester bonds in BDNPP, generating the monoester DNPP and inorganic phosphate in the process. In contrast, DNPP is not a substrate for 3. The crystal structure of the complex formed by 3 and DNPP reveals the formation of a tetranuclear zinc complex [Zn(TPPNOL)(DNPP)](ClO), 4, in which the phosphate moiety of DNPP adopts an unusual tridentate μ-η:η:η coordination mode.

Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here we report the development of RhoBAST:SpyRho, a fluorescent light-up aptamer (FLAP) system ideally suited for visualizing RNAs in live or fixed cells with various advanced fluorescence microscopy modalities. Overcoming problems associated with low cell permeability, brightness, fluorogenicity, and signal-to-background ratio of previous fluorophores, we design a novel probe, SpyRho (Spirocyclic Rhodamine), which tightly binds to the RhoBAST aptamer. High brightness and fluorogenicity is achieved by shifting the equilibrium between spirolactam and quinoid. With its high affinity and fast ligand exchange, RhoBAST:SpyRho is a superb system for both super-resolution SMLM and STED imaging. Its excellent performance in SMLM and the first reported super-resolved STED images of specifically labeled RNA in live mammalian cells represent significant advances over other FLAPs. The versatility of RhoBAST:SpyRho is further demonstrated by imaging endogenous chromosomal loci and proteins.