UC San Diego

The potential of thienoguanosine (^thG), an isomorphic fluorescent analogue of guanosine, was evaluated as a reporter for probing the structure and dynamics of hTel22, a human telomeric G-quadruplex (G4) sequence. Nine of the 12 guanines directly participating in G-tetrads were individually substituted with ^thG, and their impact on the hTel22 structure was examined by circular dichroism, thermal melting, and ¹H NMR spectroscopy. In Na⁺ buffer, substitutions at external tetrads maintained the native antiparallel topology and thermal stability, while substitutions within the middle tetrad significantly disrupted the G4 structure. Molecular dynamics simulations supported that ^thG incorporation in external tetrads is well tolerated, whereas it alters base stacking and Na⁺ coordination in middle tetrad positions. In K⁺ buffer, external substitutions favored the antiparallel over the hybrid topology, resulting in a modest destabilization. ^thG-labeled sequences at external tetrads in both Na⁺ and K⁺ buffers exhibited high fluorescence quantum yields, long fluorescence lifetimes, and high sensitivity to local conformation. As an application, ^thG fluorescence signal proved instrumental in accurately characterizing the kinetics of Na⁺-induced G4 folding, enabling all kinetically resolved folding steps to be captured from a single fluorescence observable. Overall, ^thG serves as a remarkably sensitive, minimally perturbing fluorescent guanosine analogue for structural and dynamic studies of G4s. Moreover, its longest lifetime (18.5 to 28.5 ns), at least twice that in DNA duplexes, is suitable for time-gated detection with high signal-to-noise ratio and easy distinction of G4s from duplexes, offering a key advantage for G4-targeting drug discovery.