Cellular RNA labeling using light-up aptamers that bind to and activate fluorogenic molecules has gained interest in recent years as an alternative to protein-based RNA labeling approaches. Aptamer-based systems are genetically encodable and cover the entire visible spectrum. However, the relatively weak nature of the non-covalent aptamer-fluorogen interaction limits the utility of these systems in that multiple copies of the aptamer are often required, and in most cases the aptamer must be expressed on a second scaffold such as a transfer RNA. We propose that these limitations can be averted through covalent RNA labeling, and here we describe a photoaffinity approach in which the aptamer ligand is functionaliz... More
Cellular RNA labeling using light-up aptamers that bind to and activate fluorogenic molecules has gained interest in recent years as an alternative to protein-based RNA labeling approaches. Aptamer-based systems are genetically encodable and cover the entire visible spectrum. However, the relatively weak nature of the non-covalent aptamer-fluorogen interaction limits the utility of these systems in that multiple copies of the aptamer are often required, and in most cases the aptamer must be expressed on a second scaffold such as a transfer RNA. We propose that these limitations can be averted through covalent RNA labeling, and here we describe a photoaffinity approach in which the aptamer ligand is functionalized with a photoactivatable reactive group such that irradiation with UV light results in covalent attachment to the RNA of interest. In addition to the robustness of the covalent linkage, this approach benefits from the ability to temporally control RNA labeling. To demonstrate this method, we incorporated a photoaffinity linker onto malachite green and fused the malachite green aptamer to a specific mRNA reporter of interest. We observed markedly improved sensitivity for fixed cell imaging of mRNA using this approach compared to in situ hybridization. Additionally, we demonstrate visualization of RNA dynamics in live cells using an mRNA having only a single copy of the aptamer, minimizing perturbation of the structure and localization. Our initial biological application utilizes the photoaffinity labeling approach to monitor RNA stress granule dynamics and we envision future application of this method for a wide range of investigations into the cellular localization, dynamics, and protein binding properties of cellular RNAs.
