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

Abstract

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 (2F-araC) and 2-fluoro-ribocytidine (2F-riboC) have been shown to influence i-motif structures differently, with 2F-araC strongly stabilizing and 2F-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.