A Bright and Ionizable Cytosine Mimic for i-Motif Structures

Abstract

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 (λem = 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.

Fluorescent molecular rotors detect O6-methylguanine dynamics and repair in duplex DNA

“…1) that also exhibits molecular rotor properties. 26 Our MGMT binding and repair results obtained using ts C-containing duplexes ts C:MG (Fig. S8–S11, ESI†) mirror those obtained using ts T (Fig.…”

“…Such dye–quencher FRET systems are highly sensitive, but they require multiple and bulky substrate modifications – for example with fluorescein and Dabcyl, 15 and they offer little-to-no spatial resolution under 25 Å due to their typical Förster radius ( R o ) of ∼50 Å. 17 In contrast, fluorescent nucleobase analogues (FBAs) 18,19 require only a single site of label incorporation and can probe ribozyme activity, 20 protein binding, 19,21,22 enzymatic transformations, 23 metal binding, 24 and DNA conformational changes 25,26 in real time with single-nucleotide spatial resolution. For biological applications, DNA and RNA polymerases can recognize FBAs that mimic Watson–Crick base pairs with high fidelity for incorporation into RNA and DNA molecules in vitro 27–31 and in vivo .…”