Investigating DNA-Mediated Charge Transport by Time-Resolved Spectroscopy

The complex [Re(CO)3(dppz)(py-OR)]+ (dppz = dipyrido[3,2-a:2',3'-c]-phenazine; py'-OR = 4-functionalized pyridine) offers IR sensitivity and can oxidize DNA directly from the excited state. The behavior of several covalent and noncovalent Re-DNA constructs was monitored by time-resolved IR (TRIR) and UV/visible spectroscopies, as well as biochemical methods, confirming the ability of the complex to trigger long-range oxidation of DNA. Optical excitation of the complex leads to population of metal-to-ligand charge transfer excited states and at least two distinct intraligand charge transfer excited states. Several experimental observations are consistent with charge injection by excited Re*. These include similarity between TRIR spectra and the spectrum of reduced Re observed by spectroelectrochemistry, the appearance of a guanine radical signal in TRIR spectra, and the eventual formation of permanent guanine oxidation products. The majority of reactivity occurs on the ultrafast time scale, although processes dependent on slower conformational motions of DNA, such as the accumulation of oxidative damage at guanine, are also observed.;The photooxidation activity of this Re complex was compared directly to that of other metallointercalators that have been used previously in our laboratory to oxidize DNA. The complexes [Rh(phi)2(bpy')]3+ (phi = 9,10-phenanthrenequinone diimine; bpy' = 4-methyl-4'-(butyric acid)-2,2'-bipyridine), [Ir(ppy)2(dppz')]+ (ppy = 2-phenylpyridine; dppz' = 6-(dipyrido[3,2- a:2',3'-c]phenazin-11-yl)hex-5-ynoic acid), and [Re(CO)3(dppz)(py'-OH)]+ (py'-OH = 3-(pyridin-4-yl)-propanoic acid) were each covalently tethered to DNA. Biochemical studies show that upon irradiation, the three complexes oxidize guanine by long-range DNA-mediated CT with the efficiency: Rh > Re > Ir. Comparison of spectra obtained by spectroelectrochemistry after bulk reduction of the free metal complexes with those obtained by transient absorption (TA) spectroscopy of the conjugates suggests that excitation of the conjugates at 355 nm results in the formation of the reduced metal states. Electrochemical experiments and kinetic analysis of the TA decays verify that the primary factors responsible for the trend observed in the guanine oxidation yield of the three complexes are the thermodynamic driving force for CT, variations in the efficiency of back electron transfer, and coupling to DNA.;The ability of redox-active DNA-binding proteins to act as hole sinks in DNA-mediated CT systems was also studied by time-resolved spectroscopy. Such experiments are designed to provide support for the utilization of DNA-mediated CT in biological systems. In studies involving the cell cycle regulator p53, photoexcitation results in the formation of a weak transient band at 405 nm. This band, which is not observed in samples lacking the protein, resembles the primary spectral feature of the tyrosine cation radical. Although the signal is weak and reproducibility is inconsistent, these results suggest that photolysis of the sample leads to DNA-mediated oxidation of tyrosine in p53. Similar experiments were conducted on the transcriptional activator SoxR. Here, the presence of dithionite, required in solution to keep the protein reduced, complicates the photochemistry of the system considerably. Regardless, a weak absorbance at 418 nm that develops following photolysis at 355 nm provides evidence for the DNA-mediated oxidation of the protein. The behavior of the base excision repair protein endonuclease III was also observed in the presence of DNA and metal complex oxidants. In flash-quench studies, addition of the protein results in the formation of a strong negative signal at 410 nm in TA traces. In studies involving direct photooxidation by Rh, Ir, and Re complexes, no new transients are detected upon the addition of protein, but changes in the intensities of the resultant TA spectra and in the steady-state absorbance spectra following photolysis indicate that DNA-mediated oxidation of the protein may be taking place. (Abstract shortened by UMI.).