Environment-Sensitive Nucleoside Probe Unravels the Complex Structural Dynamics of i-Motif DNAs

Although evidence for the existence and biological role of i-motif (iM) DNA structures in cells is emerging, probing their structural polymorphism and identifying physiologically active conformations using currently available tools remain a major challenge. Here, we describe the development of an innovative device to investigate the conformation equilibrium of different iMs formed by C-rich telomeric repeat and oncogenic Braf promoter sequences using a new conformation-sensitive dualpurpose nucleoside probe. The nucleoside is composed of a trifluoromethyl-benzofuran-2-yl moiety at the CS position of 2'-deoxyuridine, which functions as a responsive fluorescent and F-19 NMR probe. While the fluorescent component is useful in monitoring and estimating the folding process, the F-19 label provides spectral signatures for various iMs, thereby enabling a systematic analysis of their complex population equilibrium under different conditions (e.g., pH, temperature, metal ions, and cell lysate). Distinct F-19 signals exhibited by the iMs formed by the human telomeric repeat helped in calculating their relative population. A battery of fluorescence and F-19 NMR studies using native and mutated B-raf oligonucleotides gave valuable insights into the iM structure landscape and its dependence on environmental conditions and also helped in predicting the structure of the major iM conformation. Overall, our findings indicate that the probe is highly suitable for studying complex nucleic acid systems.