Programmable, Structure-Switching RhoBAST for Hybridization-Mediated mRNA Imaging in Living Cells.

The development of fluorescent probes for visualizing endogenous RNAs in living cells is crucial to understand their complex biochemical roles. Recently, we developed RhoBAST, one of the most photostable and brightest fluorescence light-up aptamers (FLAPs), as a genetically encoded tag for imaging messenger RNAs (mRNAs). Here, we describe programmable RhoBAST sequences flanked by target-binding hybridization arms that light up only when bound to the untagged target RNA in trans. As part of the hybridization arm, we introduced a modular transducer sequence that switches the secondary structure of RhoBAST and renders it incapable of binding to its fluorogenic ligand TMR-DN. Only the specific binding of the hybridization arms to the target RNA triggers the correct folding of RhoBAST and fluorescence light-up after binding to TMR-DN. We characterized the structural switching of programmable RhoBAST sequences extensively in vitro and applied them to visualize untagged mRNAs in live bacteria.

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

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.

Avidity-based bright and photostable light-up aptamers for single-molecule mRNA imaging.

Fluorescent light-up aptamers (FLAPs) have emerged as valuable tools to visualize RNAs, but are mostly limited by their poor brightness, low photostability, and high fluorescence background in live cells. Exploiting the avidity concept, here we present two of the brightest FLAPs with the strongest aptamer-dye interaction, high fluorogenicity, and remarkable photostability. They consist of dimeric fluorophore-binding aptamers (biRhoBAST and biSiRA) embedded in an RNA scaffold and their bivalent fluorophore ligands (bivalent tetramethylrhodamine TMR2 and silicon rhodamine SiR2). Red fluorescent biRhoBAST-TMR2 and near-infrared fluorescent biSiRA-SiR2 are orthogonal to each other, facilitating simultaneous visualization of two different RNA species in live cells. One copy of biRhoBAST allows for simple and robust mRNA imaging with strikingly higher signal-to-background ratios than other FLAPs. Moreover, eight biRhoBAST repeats enable single-molecule mRNA imaging and tracking with minimal perturbation of their localization, translation, and degradation, demonstrating the potential of avidity-enhanced FLAPs for imaging RNA dynamics.

(Oligo-)Thiophene Functionalized Tetraazaperopyrenes: Donor-Acceptor Dyes and Ambipolar Organic Semiconductors.

Tetraazaperopyrenes (TAPPs) have been functionalized with thiophene and terthiophene units of different architecture resulting in a variety of organic donor-acceptor (D-A) compounds. The influence of the connection of the thiophenes to the TAPP core on their structural, photophysical and electrochemical properties has been studied in detail by a combination of X-ray crystallography, UV-vis and fluorescence spectroscopy as well as cyclic voltammetry, which allowed the establishment of structure-property relationships. The HOMO-LUMO gap is significantly decreased upon substitution of the TAPP core with electron-donating thiophene units, the extent of which is strongly influenced by the orientation of the thiophene units. The latter also crucially directs the molecular packing in the solid. Linkage at the α-position allows both inter- and intramolecular N···S interaction, whereas linkage in the ß-position prevents intramolecular N···S interaction, resulting in a less pronounced conjugation of the TAPP core and the thiophene units. The new TAPP derivatives were processed as semiconductors in organic thin-film transistors (TFTs) that show ambipolar behavior. The insight into band gap and structure engineering may open up new possibilities to tailor the electronic properties of TAPP-based materials for certain desired applications.