Talk on Friday 02:15-02:30pm submitted by Sydney Willey
Fluorogenic U-rich internal loop (FLURIL) tagging with bPNA enables intracellular RNA and DNA tracking
Yufeng Liang (Department of Chemistry and Biochemistry, The Ohio State University), Sydney Willey (Department of Biological Chemistry and Pharmacology, The Ohio State University), Yu-Chieh Chung (Department of Biological Chemistry and Pharmacology, The Ohio State University), Yi-Meng Lo, Shiqin Miao, Sarah Rundell (Department of Chemistry and Biochemistry, The Ohio State University), Li-Chun Tu (Department of Biological Chemistry and Pharmacology, The Ohio State University), Dennis Bong (Department of Chemistry and Biochemistry, The Ohio State University)
RNA and DNA are highly dynamic and provide complex, essential regulation to cells. Fluorescent methods for intracellular RNA imaging and tracking in living cells are crucial for understanding RNA and DNA function and organization. While many useful fluorescent tools exist, they often require multiple binding sites and large proteins to reach a sufficient signal-to-noise ratio, which can potentially impact the structure and function of the target of interest. In this research, we developed a novel method for intracellular RNA and DNA tracking: fluorogenic U-rich internal loop (FLURIL) tagging. Fluorophore-labeled bifacial peptide nucleic acids (fbPNAs) fluorescently brighten when forming base-triple hybrid stems specifically within genetically encoded U4xU4 U-rich internal loops in RNA. The cell-permeable fbPNA (~1 kD) binds with minimal structural modifications, and does not require RNA-binding proteins or fluorescent proteins. Using FLURIL tagging of the bacteriophage MS2 hairpin in fixed HEK-293 cells, we successfully visualized its binding with MS2 coat protein (MCP). Next, we visualized a native mammalian ribonucleoprotein (RNP) complex by FLURIL tagging of RNA containing UG repeats which bind to transactive response DNA binding protein (TDP-43). In addition, we tested FLURIL live-cell tracking by targeting guide RNA (gRNA) in a CRISPR-based imaging system to visualize and track a genomic locus in live U2OS cells. Compared with CRISPR-Sirius, which uses MS2/MCP-HaloTag labeled gRNA for live-cell genomic loci tracking, FLURIL had a similar brightness while adding significantly less steric bulk to the target locus. Overall, FLURIL tagging effectively tracks RNA, RNPs, and genomic loci and can be applied to living cells in real time. By minimally altering the native structure of RNA, FLURIL tagging allows accurate tracking of the structure and function of RNA which could improve studies in diseased cells and potentially lead to the development of new treatments.
Keywords: RNA-based fluorescent tagging, Imaging RNA and DNA tracking, live-cell imaging