Inside the living cell is the molecular crowding environment, characterized by high concentrations of proteins, nucleic acids, and polysaccharides. It has been documented that the structure, thermodynamic properties, and functionality of nucleic acids in such environments differ significantly from those in dilute solutions. While the relationship among the structure, base-pair opening dynamics, and the binding with DNA-binding proteins of DNA in dilute solutions has been established, understanding the corresponding structural dynamics within the molecular crowding environment remains elusive. Notably, a non-canonical DNA structure known as the DNA triplex has been linked to Friedreich's ataxia. In our previous work, we provided direct evidence of DNA triplex formation within living human cells employing in-cell NMR techniques [1-5]. Here, we explore the dynamics of the base pairs involved in the DNA triplex structure under molecular crowding conditions through NMR relaxation dispersion experiments and water magnetization transfer experiments. By employing NMR analysis, we successfully determine the imino proton-water proton exchange rates, as well as the sum of the base-pair opening and closing rate constants (kex) of the base pairs within the DNA triplex structure, both in dilute solution and within molecular crowding environments. Our findings demonstrate distinct dynamics of base pairs within the DNA triplex structure under molecular crowding conditions compared to those observed in dilute solutions.
1) Y. Yamaoki, T. Nagata, T. Sakamoto, M. Katahira, Biophys. Rev., 2020, 12, 411-417
2) Y. Yamaoki, T. Nagata, K. Kondo, T. Sakamoto, S. Takami, M. Katahira, Nat. Commun., 2022, 13, 7143
3) O. Eladl, Y. Yamaoki, K. Kondo, T. Nagata, M. Katahira, Chem. Commun., 2022, 22, 102-105
4) T. Sakamoto, Y. Yamaoki, T. Nagata, M. Katahira, Chem. Commun., 2021, 57, 6364-6367
5) O. Eladl, Y. Yamaoki, K. Kondo, T. Nagata, M. Katahira, Int. J. Mol. Sci., 2023, 24, 9069