A Bright and Ionizable Cytosine Mimic for -Motif Structures.

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