Developing robust methods to monitor non-canonical nucleic acid conformational equilibrium and their interactions with drug-like ligand molecules is essential for exploring the functional paradigm of nucleic acids and facilitating the small-molecule drug discovery against bioactive nucleic acid targets. Due to its high natural abundance, sensitivity, chemical shift dispersion, biocompatibility, and straightforward spectrum pattern, 19F NMR has become incredibly attractive for investigating biomolecular interaction. This project aimed to utilize FANA mutation (replacing H2' atom of sugar by 19F) as a robust site-specific 19F labeling strategy on G-quadruplex, i-Motif forming DNA sequences to exploit their structural equilibrium, dynamics, and ligand binding interactome in vitro and the intracellular space. This project will combine 19F NMR and complementary native mass spectrometry, as well as low-resolution spectroscopic assays, to give fundamental insights into the dynamic structural interconversion of non-canonical nucleic acid-based regulatory and signaling pathways.