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)..

Although genetically encoded light-up RNA aptamers have become promising tools for visualizing and tracking RNAs in living cells, aptamer/ligand pairs that emit in the far-red and near-infrared (NIR) regions are still rare. In this work, we developed a light-up RNA aptamer that binds silicon rhodamines (SiRs). SiRs are photostable, NIR-emitting fluorophores that change their open–closed equilibrium between the noncolored spirolactone and the fluorescent zwitterion in response to their environment. This property is responsible for their high cell permeability and fluorogenic behavior. Aptamers binding to SiR were in vitro selected from a combinatorial RNA library. Sequencing, bioinformatic analysis, truncation, and mutational studies revealed a 50-nucleotide minimal aptamer, SiRA, which binds with nanomolar affinity to the target SiR. In addition to silicon rhodamines, SiRA binds structurally related rhodamines and carborhodamines, making it a versatile tool spanning the far-red region of the spectrum. Photophysical characterization showed that SiRA is remarkably resistant to photobleaching and constitutes the brightest far-red light-up aptamer system known to date owing to its favorable features: a fluorescence quantum yield of 0.98 and an extinction coefficient of 86 000 M–1cm–1. Using the SiRA system, we visualized the expression of RNAs in bacteria in no-wash live-cell imaging experiments and also report stimulated emission depletion (STED) super-resolution microscopy images of aptamer-based, fluorescently labeled mRNA in live cells. This work represents, to our knowledge, the first application of the popular SiR dyes and of intramolecular spirocyclization as a means of background reduction in the field of aptamer-based RNA imaging. We anticipate a high potential for this novel RNA labeling tool to address biological questions.

In order to gain deeper insight into the functions and dynamics of RNA in cells, the development of methods for imaging multiple RNAs simultaneously is of paramount importance. Here, we describe a modular approach to image RNA in living cells using an RNA aptamer that binds to dinitroaniline, an efficient general contact quencher. Dinitroaniline quenches the fluorescence of different fluorophores when directly conjugated to them via ethylene glycol linkers by forming a non-fluorescent intramolecular complex. Since the binding of the RNA aptamer to the quencher destroys the fluorophore-quencher complex, fluorescence increases dramatically upon binding. Using this principle, a series of fluorophores were turned into fluorescent turn-on probes by conjugating them to dinitroaniline. These probes ranged from fluorescein-dinitroaniline (green) to TexasRed-dinitroaniline (red) spanning across the visible spectrum. The dinitroaniline-binding aptamer (DNB) was generated by in vitro selection, and was found to bind all probes, leading to fluorescence increase in vitro and in living cells. When expressed in E. coli, the DNB aptamer could be labelled and visualized with different-coloured fluorophores and therefore it can be used as a genetically encoded tag to image target RNAs. Furthermore, combining contact-quenched fluorogenic probes with orthogonal DNB (the quencher-binding RNA aptamer) and SRB-2 aptamers (a fluorophore-binding RNA aptamer) allowed dual-colour imaging of two different fluorescence-enhancing RNA tags in living cells, opening new avenues for studying RNA co-localization and trafficking.

Copper, a key cofactor for many life processes, is toxic at elevated levels, and its availability is strictly controlled inside cells. Therefore, it is a challenge to visualize copper availability in the tight copper-binding environment of the cell. We report a genetically encoded fluorescent copper(I) sensor based on the copper(I)-binding-induced conformational change of a copper-responsive transcriptional regulator, Amt1. The resulting reporter, Amt1-FRET, is ratiometric, highly sensitive (K(d) = 2.5 x 10(-18) M), and selective toward copper(I). Its measured high affinity to copper(I) confirms the extremely low copper availability in yeast since Amt1 senses the upper limit of cellular copper levels in yeast and activates copper detoxification genes. Amt1-FRET operates in the dynamic range of the cellular copper buffer in mammalian cells and can report dynamic fluctuations of the cellular copper availability within minutes of perturbation. Thus, Amt1-FRET visualizes the tightly controlled copper availability in mammalian cells.

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