Detecting RNA dynamics in live mammalian cells with fluorescence lifetime-based sensors

RNA function is closely linked with subcellular localization and interaction with other biological molecules such as other RNAs and proteins. Acute cellular perturbations and disease mutations may alter RNA dynamics and hence function. Therefore, visualizing RNAs in live cells is critically important for a mechanistic understanding. However, robust fluorescence labels for specific RNA molecules to achieve quantitative detection in the complex cellular environment for diverse RNA species are under-developed. Typically, fluorescent RNA tags are engineered where a genetically encoded short RNA binds a small molecule that conveys fluorescence properties. These efforts aim to generate large fluorescence intensity changes of the fluorophore upon binding the RNA. Here, we use fluorescence lifetime as an orthogonal approach for a fluorescence RNA sensor. We used the Riboglow platform, an RNA tagging platform that relies on binding of a small fluorescent molecule to a short RNA sequence. First, we compared probe fluorescence signal in live cells in the presence and absence of the RNA tag. We found that fluorescence lifetime provides substantially improved contrast compared with fluorescence intensity. Second, we assessed sensitivity of monitoring subcellular RNA localization dynamics live via fluorescence lifetime. Third, we are exploring modularity of the system by varying the RNA- and probe component to enable multi-parameter RNA detection. Together, we find that fluorescence lifetime offers advantages to track RNAs and quantify localization versus fluorescence intensity. We demonstrated the versatility of this system in diverse cellular examples. Our system is the first platform that exploits fluorescence lifetime for live RNA tracking in cells. We plan to expand this imaging modality further to quantitatively assess RNA dynamics. © FASEB.