Trafficking of messenger RNA (mRNA) to subcellular compartments plays an essential role in RNA homeostasis and cellular function. This spatiotemporal control of mRNA localization is a common characteristic for a significant fraction of transcripts, and in recent years, fluorescent labeling and imaging of RNA has dramatically increased our understanding of the heterogeneity of transcript regulation and the complex subcellular interactions of RNAs and proteins. A number of non-covalent methods have been explored to label specific transcripts in living cells, and most recently, the small molecule binding capabilities of RNA have been harnessed for this purpose through the development of aptamers that bind to fluorogenic dyes. However, these approaches generally require multiple copies of the aptamer to be fused to the RNA of interest, potentially perturbing its structure, localization, and function.<br/><br/>We hypothesized that the limitations of the current RNA labeling approaches could be overcome through covalent attachment of the fluorescent molecule to the target RNA. Specifically, we envisioned a photoaffinity labeling approach in which a fluorogenic dye is functionalized with a photoreactive diazirine group such that UV irradiation results in covalent labeling of the RNA of interest. For our initial demonstration of this approach, we used the previously reported malachite green (MG) aptamer, as the MG dye benefits from far red shifted fluorescence emission and thus is well-suited for cellular imaging. Using our photoaffinity approach, we demonstrate that an mRNA of interest can be labeled and imaged in both fixed and live cells using a single 57 nt fusion. This is significantly smaller than the fusions required in other aptamer-based methods, minimizing perturbation of RNA structure and localization. We demonstrate that covalent labeling enables RNA visualization under conditions where the non-covalent system fails, and we utilize our approach to monitor the dynamics of RNAs in stress granules. Together, this research introduces the first covalent method for cellular RNA labeling and provides an effective and easy-to-use tool for the bioconjugation community.

Imaging and tracking mRNA in live mammalian cells via fluorogenic photoaffinity labeling

ACS Fall 2020 Virtual Meeting

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