Understanding and optimization of the photophysics of fluorescent nucleoside analogues are critical for their applications in probing the structure and dynamics of nucleic acids, and studying their interactions with ligands and biomolecules.
Thienoguanosine ( th G) is an isomorphic guanosine (G) surrogate that almost perfectly mimics G in nucleic acids. To exploit its full potential and lay the foundation for future applications, twenty DNA duplexes, where the bases facing and neighboring th G were systematically varied, were thoroughly studied using fluorescence spectroscopy, molecular dynamics simulations and mixed quantum mechanical/molecular mechanics calculations, yielding a comprehensive understanding of its photophysics in DNA. In matched duplexes, th G's hypochromism was larger for flanking G/C residues but its fluorescence quantum yield (QY) and lifetime values were almost independent of the flanking bases. This was attributed to high duplex stability, which maintains a steady orientation and distance between nucleobases, so that a similar charge transfer (CT) mechanism governs the photophysics of th G independently of its flanking nucleobases. th G can therefore replace any G residue in matched duplexes, while always maintaining similar photophysical features. In contrast, the local destabilization induced by a mismatch or an abasic site restores a strong dependence of th G's QY and lifetime values on its environmental context, depending on the CT route efficiency and solvent exposure of th G. Due to this exquisite sensitivity, th G appears ideal for monitoring local structural changes and single nucleotide polymorphism.Moreover, th G's dominant fluorescence lifetime in DNA is unusually long (9-29 ns), facilitating its selective measurement in complex media using a lifetime-based or a time-gated detection *
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