Development of reactions for the five prime fluorescent tagging of RNA to visualize structural conformations

The 3-dimensional conformations of RNA molecules are crucial to understanding their regulatory functions in gene expression and biological processes. Despite advancements in RNA 3D structural elucidation through fluorescent probing, computational methods, and traditional characterization methods such as NMR and X-ray diffraction, differentiating between G-quadruplex (G4) and hairpin loop RNA conformations remains a significant challenge. These conformations have distinct implications for microRNA gene regulation, and effective structural differentiation could enhance our understanding of RNA's role in various diseases and advance conformation selective micro-RNA based drug discovery. This project aims to develop efficient chemical reactions for attaching a fluorescent probe to the 5' end of RNA that can be used in a fluorescence resonance energy transfer (FRET) system to observe RNA structure. We have modified the 5' alcohol of adenosine using iodination, azidation, esterification, and oxidation reactions. By synthesizing 5'-azido-5'-deoxyadenosine and 5,6 -propargylamidofluorescein, we have observed that conjugation can be achieved by a copper-catalyzed alkyne azide click reaction. Ongoing work involves altering the synthetic route of the azide modification on the RNA model to improve reaction efficiency. Other direct esterification and amine coupling methods using 5(6)-carboxyfluorescein and 5-aminofluorescein have been attempted to achieve conjugation to the RNA model. Future directions will involve improving the efficiency and compatibility of our reactions to attach a fluorophore directly to RNA. The resulting fluorophore-modified RNA will be visualized using fluorescence-based assays, allowing for differentiation between G-quadruplex and hairpin loop forms.