NO PROTONS WERE HARMED IN THE MAKING OF THIS THESIS: SELECTIVE 15N AND 2H ISOTOPIC LABELING AND NMR RELAXATION EXPERIMENTS

Abstract

The diverse roles RNA (ribonucleic acid) play in cellular processes are fundamentally linked to their unique architectures and dynamic motions. Even subtle changes in RNA sequence, structure, or environment can impact their stability and biological functions. Solution Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for high-resolution structural and dynamic analysis. However, applying NMR spectroscopy to large RNAs remains challenging. The glutamine riboswitch, a key regulatory RNA element controlling gene expression in response to glutamine levels, comprises a structured aptamer domain (63 nucleotides) and full-length aptamer + ribosome binding site (RBS) (100 nucleotides), enabling intricate control of translation initiation. However, solution Nuclear Magnetic Resonance (NMR) spectroscopy faces severe challenges in analyzing such large RNAs due to limited chemical shift dispersion and pronounced line broadening effects, which severely restrict studies to smaller RNA fragments, typically under 60 nucleotides. To overcome these barriers, selective isotopic labeling strategies have emerged as critical tools, enhancing spectral resolution and sensitivity by reducing spectral crowding and improving relaxation properties. In particular, the application of atom-specific labeling combined with antiphase coherence and direct 15N detection offers promising alternatives for studying larger RNAs by mitigating relaxation losses of chemical shift information. Here, we apply these advanced NMR approaches to investigate several RNAs ranging from 14 to 100 nucleotides. Our results highlight the potential for these methods to push the boundaries of NMR spectroscopy, extending its applicability to a broader range of large and functionally important RNAs.