A Highly Fluorescent Nucleobase Molecular Rotor.

Fluorescent base analogs (FBAs) are powerful probes of nucleic acids' structures and dynamics. However, previously reported FBAs exhibit relatively low brightness and therefore limited sensitivity of detection. Here we report the hitherto brightest FBA that has ideal molecular rotor properties for detecting local dynamic motions associated with base pair mismatches. The new trans-stilbene annulated uracil derivative "tsT" exhibits bright fluorescence emissions in various solvents (Ԑ × Φ = 3,400 - 29,700 cm-1 M-1) and is highly sensitive to mechanical motions in duplex DNA (Ԑ × Φ = 150 - 4,250 cm-1 M-1). tsT is thereby a "smart" thymidine analog, exhibiting a 28-fold brighter fluorescence intensity when base paired with A as compared to T or C. Time-correlated single photon counting revealed that the fluorescence lifetime of tsT (τ = 4 - 11 ns) was shorter than its anisotropy decay in well-matched duplex DNA (θ = 20 ns), yet longer than the dynamic motions of base pair mismatches (0.1 - 10 ns). These properties enable unprecedented sensitivity in detecting local dynamics of nucleic acids.

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