Ionized nucleobases participate in pairing interactions outside of Watson and Cricks rules. Base pairing and ionization can be coupled via global conformational changes to raise the apparent pKa of protonated nucleobases to values above physiological pH. To provide the first specific reporter of proton-coupled DNA folding, we developed a push-pull fluorescent nucleoside analog composed of dimethylaniline (DMA) fused to deoxycytidine. C-DMA exhibits the same pKa and base pairing characteristics as native cytosine residues in the human telomeric repeat sequence, where it causes little or no perturbation of DNA structure or stability. Upon protonation of C-DMA, enhanced charge transfer results in large red shifting (+40 nm) of its excitation/emission maxima. Cs-DMA fluorescence intensity, anisotropy, and energy transfer properties can be used to track conformational changes in real time. Strand displacement assays were conducted by mixing C-DMA-labeled duplexes containing a 5' single-stranded overhang with an excess of unlabeled DNA to initiate thermodynamically favorable unfolding-refolding reactions that release the C-DMA-labeled strand from its complement. Rate constants for strand displacement upon addition of i-motif DNA (k = 1.0 M-(1) s-(1), t(1/2) approximate to 12 h) were 320-fold lower than those measured upon addition of unfolded DNA (k = 3.2 x 102 M-(1) s-(1), t(1/2) approximate to 2 min). These results reveal that i-motif structures having only marginal thermodynamic stabilities (T-m < 40 degrees C) can still pose large kinetic barriers to duplex formation under near-physiological conditions of pH (5.75), temperature (25 degrees C), and salt (100 mM NaCl).