| Nucleic acids are highly dynamic and are not present as a single rigid structure. For RNA molecules, the structure, energetics and dynamics are closely connected to their cellular functions, therefore it is very important to characterize these interactions. Conventional biophysical methods such as X-ray crystallography and NMR are highly powerful tools in deriving the structures of RNA molecules at the atomic level. However, structural conformational dynamics are difficult to probe due to rapid structural inter-conversion. Using the theophylline-binding RNA aptamer and transcription antiterminator N protein/boxB RNA complex as two different model systems, here we employed femtosecond time-resolved spectroscopy as a main tool, using 2-aminopurine as a probe to capture the dynamic base stacking interactions. In the theophylline-binding RNA aptamer, we found that some regions of the aptamers are conformationally heterogeneous in the free state, while other regions are pre-organized in their bound-state conformation. In the boxB RNA, we proposed a revised model for the boxB RNA/N peptide interface which consists of three different patterns of states with stacking/unstacking interaction. We also achieved an increase in the stacking population by using new RNA binding ligands. In conclusion, we observed RNA as an ensemble of structures in different structural contexts rather than a single rigid static structure. We proposed a detailed model for free RNA and RNA/ligand complexes using the femtosecond time-resolved spectroscopy that is complementary to these from conventional biophysical methods such as NMR and X-ray crystallography. |
