Design and photophysical characterization of quasi-intrinsic fluorescent probes utilized in DNA sequencing

To understand the gene-based biological processes in-depth, the single-molecule real-time sequencing has drawn increasing attention with promoted by the Human Genome Project. Herein, a set of newly designed canonical fluorescent bases (A_y, tC, G_b, T_p) are proposed for four-color DNA sequencing. These quasi-intrinsic probes are derived from the fluorophore replacement and ring expansion on natural bases, which still keep the pyrimidine or purine underlying skeleton and Watson–Crick hydrogen bonding face to allow minimal perturbation to the native DNA duplex. More importantly, these nucleobase analogues possess red-shifted absorption and efficient photoluminescence due to the enhanced π-conjugation in character. Meanwhile, the four analogues could generate distinct emission wavelength (Δλ ∼ 50 nm) for real-time sequencing. To assess the biological employment of the proposed biosensors, the effects of base pairing and linking deoxyribose are also considered.