UC San Diego

Nucleic acids have been shown to have roles beyond carrying and delivering genetic information. They have drawn interest and gained popularity as powerful tools, being developed for furthering scientific research and enabling advancements in biotechnology. The use of nucleic acids in the advancement of technologies that enable visualization, tracking, and manipulation of biological macromolecules has significantly broadened their investigation, especially RNA. The growing interest towards RNA biology due to its wide-ranging functions and impacts in the cell has been a motivator for the development of robust strategies to further study and harness its programmability. The focus of this work is to develop a strategy to generate fully encodable, robust, site-specific, covalent RNA-protein conjugates using tRNA guanine transglycosylase (TGT) from E. coli. A key step in the mechanism of guanine exchange by TGT is the formation of a covalent intermediate between TGT and its RNA substrate. It has been shown that by exchanging aspartate 264 for glutamate in the active site of TGT, a stabilized catalytic intermediate can be formed with its 25 nucleotide RNA substrate (TAG3). In this work, we demonstrate covalent complex formation with longer TAG3-containing RNA substrates in vitro and visualize them via denaturing urea-PAGE gel shift analysis. Furthermore, these covalent complexes are stable in the presence of relevant biomolecules, remaining largely intact in the presence of excess guanine and queuine and are only partially disrupted by excess preQ1. With demonstrated robustness of covalent conjugate formation in vitro, we next formed TGT-RNA conjugates in mammalian cells and visualized their localization via fluorescence microscopy. The images revealed trapping of NLS-tagged GFP-TGT in the nucleus and controlled export to the cytoplasm with the inclusion of RNA containing TAG3. Further analysis using cytoplasmic-to-nuclear fluorescence intensity ratio (CNI) revealed greater nuclear export of RNA-TGT(D264E) conjugates relative to that of wild-type TGT conjugates. Furthermore, we showed that cellular covalent TGT-RNA complexes are stable under denaturing conditions by analyzing them via immunoblotting. Together, these results establish a foundation for TGT as a highly site-selective, effective covalent RNA-protein conjugation platform for use in complex biological environments.