A Bright and Ionizable Cytosine Mimic for i -Motif Structures.

A new fluorescent cytosine analog " ts C" containing a trans -stilbene moiety was synthesized and incorporated into hemiprotonated base pairs that comprise i -motif structures. Unlike previously reported fluorescent base analogs, ts C mimics the acid-base properties of cytosine (p K a ≈ 4.3) while exhibiting bright (ε × Φ ≈ 1000 cm -1 M -1 ) and red-shifted fluorescence (λ em = 440 → 490 nm) upon its protonation in the water-excluded interface of ts C + :C base pairs. Ratiometric analyses of ts C emission wavelengths facilitate real-time tracking of reversible conversions between single-stranded, double-stranded, and i -motif structures derived from the human telomeric repeat sequence. Comparisons between local changes in ts C protonation with global structure changes according to circular dichroism suggest partial formation of hemiprotonated base pairs in the absence of global i -motif structures at pH = 6.0. In addition to providing a highly fluorescent and ionizable cytosine analog, these results suggest that hemiprotonated C + :C base pairs can form in partially folded single-stranded DNA in the absence of global i -motif structures.