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Electron Transfer Between Bases in Double Helical DNA
Shana O. Kelley and Jacqueline K. Barton
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Supplementary Material
Analysis of fluorescence decays and evaluation of dynamic quenching in Ae and A2 duplexes. As the decays of A
e are multiexponential, as expected for a DNA-bound probe, the fits of the fluorescence decay profiles do not yield single rate constants for the ET reaction between A
e and G. However, the integration of the decay curves for the G-containing and I-containing duplexes provides a measure of dynamic quenching that is independent of any data-fitting routine. These quenching yields are identical to those observed in steady-state measurements, permitting the calculation of
b.
Fq (3.4 Å): dynamic (TCSPC) = 0.76, overall (steady-state) = 0.85;
Fq (6.8 Å): dynamic = 0.15, overall = 0.18. Moreover, if ET rates were approximated for each component of the decay, a distance dependence equivalent to that observed based on the steady-state quantum yields was obtained.
The fluorescence decay kinetics for A2 are also multiexponential. However, the amount of lifetime quenching can again be quantitated and compared with steady-state quenching yields. This analysis provides very good agreement for the interstrand electron-transfer reaction with Fq (5.0 Å): dynamic (TCSPC) = 0.52, overall (steady-state) = 0.52; Fq (7.8 Å): dynamic = 0.45, overall = 0.46; Fq (10.8 Å): dynamic = 0.34, overall = 0.34; Fq (13.9 Å): dynamic = 0.20, overall = 0.24. These steady-state quantum yields can therefore be used for the accurate calculation of b. For the intrastrand electron-transfer reaction, the same agreement is not observed because of the significant amount of static quenching: Fq (3.4 Å): dynamic = 0.10, static = 0.83, overall = 0.93; Fq (6.8 Å): dynamic = 0.26, static = 0.49, overall = 0.72; Fq (10.2 Å): dynamic = 0.27, static = 0.20, overall = 0.47; Fq (13.6 Å): dynamic = 0.15, static = 0.08, overall = 0.21. Approximate amounts of static quenching listed here are determined from measurements of initial intensity for data sets obtained over 120-s time intervals. Therefore, becausestatic quenching is observed even at the longest distances where the quenching is significantly attenuated, these quenching yields cannot be analyzed to extract b.
Figure S-1. Stern-Volmer plots for fluorescence quenching titrations of A2 (right) and dAeTP (left) with dITP ( ), dGTP ( ), and dZTP ( ). The initial intensity (Io) of solutions containing 100 (M A2 or dAeTP, 100 mM phosphate (pH 7) compared with those with increasing concentrations of quencher (I) were monitored by steady-state emission spectroscopy at 20ºC. Excitation was performed at 335 nm for both fluorophores. Emission spectra were integrated from 350 to 520 nm for A2 and 350 to 550 nm for dAeTP. Low levels of quenching with dATP, dCTP, and dTTP (data not shown) observed were comparable with that obtained with dITP; these small amounts of quenching were insensitive to redox potential. Stern-Volmer quenching constants were calculated with lifetimes of 24 ns for dAeTP and 11 ns for A2.
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