Here, the synthesis, photophysical characterization, and application of an ew size-expanded thymine nucleoside, diox T,i sd escribed. diox T has desirable qualities as aT surrogate, including excellent quantum yield (0.36) and high environmental sensitivity.W hen incorporatedi nto singleand double-stranded DNA, diox T showede xcellent photophysical characteristics including ahighquantum yield (average 0.20), and unlike BgQ, demonstrated dependenceo n neighboring bases withouts ignificantd estabilizationof the duplex. Interestingly,t he matched base pair of adenine (A) and diox T has the unique property that it exhibits higher fluorescence than mismatched base pairs, and diox T has selfquenching effects. As one example of the possible applications of thesep romising features, single nucleoside polymorphism typing is demonstrated for discrimination of Ab y using diox T.T he results suggestt hat diox T can be used for a broad range of applications in chemical biology.[a] S.Calculations were carried out with the B3LYP density functional method and 6-31G* basis set. The HOMO energy of each nucleobase was calculated by DFT (B3LYP/6-31G*) at the optimized geometry by using the Spartan "16 program. A = 9-methyladenine, T = 1-methylthymine, G = 9-methylguanine, C = 1-methylcytosine.

Förster resonance energy transfer (FRET) has been used as a molecular ruler to monitor the distance between or within biomolecules. A distance- and orientation-dependent FRET system can be a more powerful tool to investigate biomolecules because of its higher spatial sensitivity. For this purpose, distance- and orientation-dependent FRET pairs have been developed using a DNA scaffold; however, none of them use an isomorphic thymidine analog. Herein, we report the construction of a FRET pair using a fluorescent thymidine analog, dioxT, as the donor and tC as the acceptor. The experimental FRET efficiency was calculated based on steady-state fluorescence spectra and time-resolved fluorescence (lifetime). The calculated values showed a periodical change, which suggests the sensitivity of FRET efficiency to the orientation of fluorophores. For further evaluation of our FRET pair, we compared the theoretical FRET efficiency with the experimental value and observed a relatively high consistency. This is the first color variable FRET system that used a fluorescent thymidine analog, which is expected to expand the applicability of nucleobase–analog FRET pairs.

Because of their stable orientations and their minimal interference with native DNA interactions and folding, emissive isomorphic nucleoside analogues are versatile tools for the accurate analysis of DNA structural heterogeneity....