A Bright and Ionizable Cytosine Mimic for i-Motif Structures

A new fluorescent cytosine analog "^tsC" containing a trans-stilbene moiety was synthesized and incorporated into hemiprotonated base pairs that comprise i-motif structures. Unlike previously reported fluorescent base analogs, ^tsC mimics the acid-base properties of cytosine (pK_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 ^tsC⁺:C base pairs. Ratiometric analyses of ^tsC 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 ^tsC 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.