Enhanced i-Motif Stability through Consecutive 2',2'-Difluorocytidine Incorporation

Chemical modifications of nucleic acids are widely used to tune stability and functionality in therapeutic and nanotechnological applications. Among these, fluorinated cytidine derivatives such as 2'-fluoro-arabinocytidine (2'F-araC) and 2'-fluoro-ribocytidine (2'F-riboC) have been shown to influence i-motif structures differently, with 2'F-araC strongly stabilizing and 2'F-riboC exerting a mildly deleterious effect. In this study, we investigate the impact of gemcitabine (2'-deoxy-2',2'-difluorocytidine, dFdC) on i-motif stability. dFdC exhibits small effects in single or double substituted sequences, but a pronounced stabilization when multiple consecutive residues are incorporated. Thermal and pH-dependent analyses demonstrate that sequences containing fully substituted dFdC maintain i-motif folding at neutral pH and show enhanced thermal stability. Structural insights suggest that this stabilization arises from a combination of factors, such as hyperconjugative interactions, hydrogen bonding, and dipole alignment, while the adaptable sugar conformation mitigates destabilizing minor groove contacts observed in other more rigid modifications, such as 2'-F-riboC. Cooperative interactions among adjacent dFdC residues and potential changes in hydration may play a key factor in reinforcing stability. These results highlight the unique capacity of dFdC to enhance i-motif robustness and suggest that strategically placed difluoro substitutions can be exploited to design i-motifs with improved stability, expanding their potential in biotechnology and therapeutic applications.