Femtosecond dynamics of the DNA intercalator ethidium and electron transfer with mononucleotides in water

Abstract

Ethidium (E) is a powerful probe of DNA dynamics and DNA-mediated electron transfer (ET). Molecular dynamical processes, such as solvation and orientation, are important on the time scale of ET. Here, we report studies of the femtosecond and picosecond time-resolved dynamics of E, E with 2'deoxyguanosine triphosphate (GTP) in water, and E with 7-deaza-2'-deoxyguanosine triphosphate (ZTP) in water; E undergoes ET with ZTP but not GTP. These studies elucidate the critical role of relative orientational motions of the donor-acceptor complex on ET processes in solution. For ET from ZTP to E, such motions are in fact the rate-determining step. Our results indicate that these complexes reorient before ET. The time scale for the solvation of E in water is 1 ps, and the orientational relaxation time of E is 70 ps. The impact of orientational and solvation effects on ET between E and mononucleotides must be considered in the application of E as a probe of DNA ET.

Figure 1

Arrangements for femtosecond TA, LD, and the fluorescence up-conversion (Inset) used in the experimental apparatus. OPA, optical parametric amplifier; F, filters; L, lenses; P, polarizers; PD, photodiodes; PMT, photomultiplier tube; λ₁, excitation beam for up-conversion; λ₂, probe beam for up-conversion; , half wave plate; BBO, beta barium borate up-conversion crystal; S, sample cell.

Figure 2

The up-converted fluorescence and TA of E in water (short time scans). The excitation pulse at 480 nm was used. (Left) Signals detected at the wavelength indicated. (Right) TA at the indicated probe wavelength. (Insets) Signals of pure water measured under the same experimental conditions.

Figure 3

(a and b) Fluorescence anisotropy (Right) and LD (Left) of E in water (a) and ethanol (b). (a and b) The fluorescence anisotropy E was measured with excitation at 500 nm and emission detected at 645 nm. (Left) The measured parallel (decay) and perpendicular (rise) signals. (c and d) The LD and TA of E in water (λ_exc = 500 nm; λ_probe = 585 nm and 645 nm).

Figure 4

The TA (at the magic angle) of E in water (b) and with 5 mM GTP (a), 2 mM ZTP (c), and 5 mM ZTP (d); excited at 500 nm and probed at 400 nm; see Methodology for details about the medium used, water for E, and buffer for ZTP and GTP with E.

Figure 5

The LD of E excited at 500 nm and probed at 645 nm with 10 mM ZTP (red), 10 mM GTP (blue), and pure water (black). The Inset shows the corresponding TA signals.