Fluorescent proteins have been invaluable for the detection of protein folding, localization, and protein-protein interactions in a living cell. Unfortunately, a genetically encoded sequence for RNA detection in vivo is not available. Tsien and co-workers have shown that malachite green has negligible fluorescence in solution, but when malachite green binds to an RNA aptamer the quantum yield was increased ~2,400 fold and turns on fluorescence. In this research, RNA aptamers will be evolved and selected in vivo. This is a novel technique that allows for high-throughput selection of RNA aptamers that bind fluorogenic dyes and are functional in vivo. In vivo RNA aptamer selection can be modified for selection of other RNA aptamers when using a fluorescent reporter. An RNA aptamer will be evolved to bind phenolphthalein (Phph), which is similar to fluorescein and should have spectral properties similar to green fluorescent protein. A second RNA aptamer will evolved to bind carboxymalachite green (CMG), which is similar to tetramethylrhodamine (TAMRA) and should have emission similar to red fluorescent protein. Nucleic acids chemically modified with fluorescein and TAMRA have been used extensively as a Forster resonance energy transfer (FRET) and the Phph and CMG aptamers should be a useful for a genetically encoded FRET pair. To analyze function of the RNA aptamers in vivo, an HIV based lentiviral transfection system will be used to monitor real-time fluorescence of the RNA genome and subsequent mRNA production after integration into the genome. RNA aptamers that turn on fluorescence will be useful to monitor viral life cycle, mRNA localization and lifetime, transportation of RNA molecules, and upregulation of RNA transcripts in cancer within a living cell. Fluorescent visualization of proteins within a living cell is of tremendous importance for science, but a comparable approach for RNA is lacking. RNA composes the genome of many human viruses and has altered expression in many diseases, including cancer. In this research, new RNA motifs that bind fluorogenic dyes will be created to allow real-time visualization of RNA in a living cell.
| Rodriguez, Erik A; Campbell, Robert E; Lin, John Y et al. (2017) The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends Biochem Sci 42:111-129 |
| Rodriguez, Erik A; Tran, Geraldine N; Gross, Larry A et al. (2016) A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods 13:763-9 |
| Rodriguez, Erik A; Wang, Ye; Crisp, Jessica L et al. (2016) New Dioxaborolane Chemistry Enables [(18)F]-Positron-Emitting, Fluorescent [(18)F]-Multimodality Biomolecule Generation from the Solid Phase. Bioconjug Chem 27:1390-1399 |
