Bhussry Seminar Series – “Investigating RNA Dynamics with Fluorescence Lifetime-Based Sensors in Live Mammalian Cells”

Presentation: “Investigating RNA Dynamics with Fluorescence Lifetime-Based Sensors in Live Mammalian Cells”

Speaker: Esther Braselmann, PhD
Clare Boothe Luce Assistant Professor
Chemistry Department
Georgetown University

Sponsored by the Department of Biochemistry and Molecular & Cellular Biology

Abstract:

Coding and non-coding ribonucleic acids (RNAs) play central roles in gene expression. Subcellular RNA localization and interaction with other biological molecules including other RNAs and proteins are linked with RNA function. For a mechanistic understanding of RNA function, visualizing and tracking RNA dynamics in live cells is critically important. However, robust fluorescence RNA labels to achieve quantitative detection and tracking in the complex cellular environment are under-developed. Previously, genetically encoded RNA tags that bind a small molecule to induce fluorescence intensity changes were developed. Here, we establish fluorescence lifetime as a complementary approach for a fluorescence RNA sensor. Our system consists of a fluorescent molecule that binds a genetically encoded RNA tag and induces fluorescence lifetime changes, enabling fluorescence lifetime imaging microscopy (FLIM). First, we compared probe fluorescence in live cells with and without the RNA tag. Fluorescence lifetime provides substantially improved contrast compared with fluorescence intensity. Second, we assessed aspects of our sensor platform that allow for modular RNA detection. Specifically, we determined that different RNA tags enable tracking of multiple target RNAs simultaneously. Current efforts are underway to quantify sensitivity of monitoring subcellular RNA localization dynamics live via fluorescence lifetime for coding and non-coding model RNAs. Together, we conclude that fluorescence lifetime offers advantages to track RNAs and quantify localization versus fluorescence intensity. Our system is the first platform that exploits fluorescence lifetime for live RNA tracking in cells. We plan to further expand this imaging modality to quantitatively assess RNA dynamics.