RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently tag and track RNAs in living cells. Here, we develop an RNA imaging platform using the Cobalamin (Cbl) riboswitch as an RNA aptamer and a series of probes containing Cbl as a fluorescent quencher linked to a range of fluorophores. We demonstrate fluorescence turn-on upon RNA binding and proof of concept for tracking both mRNA and small U RNA in live cells. Main textThe complex spatiotemporal dynamics of messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) affect virtually all aspects of cellular function. These RNAs associate with a large group of RNA binding proteins that dynamically modulate RNA localization, processing and function 1,2 . Such interactions govern mRNA processing, export from the nucleus, and assembly into translationally competent messages, as well as association into large macromolecular granules that are not translationally active, including P-bodies and stress granules (SGs) [3][4][5][6] . Similarly, uridine-rich small nuclear RNAs (U snRNAs, the RNA components of the spliceosome) 7 dynamically associate with protein components to comprise the functional spliceosomal complex in the nucleus 8 . During different stresses including bacterial infection, the U snRNAs along with the splicing machinery can be transiently sequestered in cytosolic foci All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/199240 doi: bioRxiv preprint first posted online Oct. 10, 2017; 3 called U-bodies 7 . Given the intricate connection between RNA localization, dynamics and function, there has been a strong push to develop tools for visualization of RNA in live cells to elucidate mechanisms underlying the mRNA and ncRNA life cycle.While there is a broad spectrum of tools to fluorescently tag proteins in live cells, far fewer approaches for live cell imaging of RNA exist. The most common system uses a series of tandem hairpins that bind an RNA-binding protein (MS2 or PP7 coat protein) coupled to a fluorescent protein (FP) [9][10][11] . This system has been used successfully to interrogate mRNA dynamics over time at the single molecule level 9,10 . However, one limitation of this system is that 12-24 copies of the aptamer are required to enhance fluorescence contrast, and the large size of the tag can perturb localization, dynamics and processing 12 of the RNA. An alternative approach involves attaching an aptamer to the RNA that directly binds a small molecular fluorogenic dye [13][14][15][16] . Recent years have seen the application of Spinach 17 , Broccoli 18 , and Mango 19 aptamers to label and track RNAs in live mammalian cells. However, all these aptamers evolved in vitro contain a G-quadruplex 20 , which has been shown to complicate RNA folding and ligand selectivity in mammalian cells [21][22...

Labeling and tracking biomolecules with fluorescent probes on the single molecule level enables quantitative insights into their dynamics in living cells. We previously developed Riboglow, a platform to label RNAs in live mammalian cells, consisting of a short RNA tag and a small organic probe that increases fluorescence upon binding RNA. Here, we demonstrate that Riboglow is capable of detecting and tracking single RNA molecules. We benchmark RNA tracking by comparing results with the established MS2 RNA tagging system. To demonstrate versatility of Riboglow, we assay translation on the single molecule level, where the translated mRNA is tagged with Riboglow and the nascent polypeptide is labeled with a fluorescent antibody. The growing effort to investigate RNA biology on the single molecule level requires sophisticated and diverse fluorescent probes for multiplexed, multi-color labeling of biomolecules of interest, and we present Riboglow as a new member in this toolbox.

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The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M -1 cm -1 ), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations -M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using sitedirected mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.