Ratiometric Detection of pH‐Induced i‐Motif Folding Based on a Dual Emissive Cytosine Analog

A dual-emissive cytosine analog (TCC), based on a 2-thienyl-3-hydroxychromone scaffold, is incorporated into oligodeoxynucleotides to monitor the folding state of DNA i-motif structures. This modified nucleobase exhibits two distinct emission bands (IN* and IT*), each responding differently to microenvironmental changes, enabling ratiometric detection. The photophysical properties of TCC are systematically characterized in various solvents and DNA contexts, including single-stranded, double-stranded, and i-motif-forming sequences. The IN*/IT* emission ratio and the wavelength of the IT* band act as robust and orthogonal reporters of hydration, base stacking, and protonation states. In fully paired duplexes, the T* band is quenched and blue-shifted, while i-motif folding results in both fluorescence enhancement and a redshift of the T* emission. Additionally, the probe distinguishes mismatched base pairs and abasic sites, offering further insights into local structural defects. Overall, this ratiometric nucleobase analog enables real-time, multiparametric monitoring of i-motif folding with high sensitivity, and holds promise for extension to other noncanonical DNA structures. The findings further establish the 3-hydroxychromone platform as a powerful tool for the rational design of fluorescent sensors targeting dynamic nucleic acid architectures.