Research Projects

The energy landscape concept helps to understand how proteins work and correlates their structure and dynamics with their function. Similarly, RNA molecules also fold into complex 3D structures and play crucial roles in cellular processes that can be explained by their energy landscape. RNA aptamers are short RNA oligonucleotides that bind to small molecules with high specificity and can adopt various conformations. RNA aptamers are involved in several critical biological applications, including drug development, understanding molecular mechanisms of biological processes, biomarker discovery, and biosensor development, to name a few. RNA aptamers can exhibit conformational heterogeneity due to their flexible nature, the presence of non-canonical base pairs, and the influence of the surrounding environment. This heterogeneity can have functional implications as different conformations can have different binding affinity or specificity for the target molecule. The binding affinity and lifetime of the bound state of RNA aptamers dictate a downstream regulatory pathway. Therefore, it is essential to understand the conformational heterogeneity and interconversion of such conformations in the presence of specific small molecule binders to design, advance, and optimize the use of RNA aptamers in diagnostic and therapeutic strategies. To achieve this, the proposal suggests using a strategy of incorporating novel fluorine-labeled nucleosides into RNA aptamer sequences at strategic positions. The nucleoside analogs are minimally perturbing and are highly conformation sensitive. The incorporation of fluorine-labeled nucleosides would allow measuring the interconversion of conformational sub-states on the RNA energy landscape by measuring conformational dynamics using 19F NMR relaxation experiments. Carr-Purcell-Meiboom-Gill (CPMG), rotating frame longitudinal relaxation (R1ρ), and chemical exchange saturation transfer (CEST) NMR experiments will be used to measure the dynamics at the ms-μs timescale. This timescale is where conformations sub-states, typically 2-3 kcal/mole higher in free energy, requiring breaking and formation of a few H-bonds, can be accessed and are responsible for many biological functions of RNAs. The effects of ligands and the surrounding environment on the energy landscape will also be studied. The proposed approach can significantly advance the understanding of RNA aptamers' structure and dynamics and lead to aptamer-based diagnostic and therapeutic strategies. The incorporation of fluorine-labeled nucleosides allows for sensitive and specific measurement of RNA aptamer dynamics without perturbing their canonical fold, making it a powerful tool for studying the energy landscape of RNA.