Final Activity Report Summary - DNA-ET (Selective oxidation and cleavage of DNA by photosensitized electron transfer)
The aim of this project was to better understand the mechanisms of photo-induced reactions in Deoxyribonucleic acid (DNA) model systems. The work concerned photochemical processes in DNA model systems induced by selective excitation of an N-(purin-6-yl)-pyridinium residue (Pyr), which was covalently linked to the nucleotide chain in a sequence-specific manner. The research results obtained for mononucleotides, dinucleotides and trinucleotides would also be discussed.
Moreover, an existing time-resolved fluorescence spectrofluorimeter based on time-correlated single-photon counting detection technique (IBH System 5000) was successfully upgraded. This modification included:
1. exchange of the excitation source to a modern, laser-based diodes (nanoLED);
2. calibration of monochromators;
3. installation and configuration of an analogue to digital (A-D) converter; and
4. software configuration.
The synthesis of the DNA model compounds containing fluorescent N-(purin-6-yl)-pyridinium residues was successfully completed and the products obtained within each synthesis step were controlled using High performance liquid chromatography (HPLC) with Ultraviolet (UV), fluorescence and Mass spectrometry (MS )detection.
Fluorescence quenching experiments were carried out using steady-state and time resolved techniques for intermolecular quenching of highly fluorescent N-(purin-6-yl)-pyridinium chloride by four DNA nucleoside monophosphates. The dynamic plots from the lifetimes were linear and their slopes were practically independent of the quencher used, while the static ones, from the fluorescence intensities, showed significant upward curvature. The latter observation was indicative of a substantial contribution of a static quenching to the overall quenching process; however, no ground state complexation could be observed when studied by UV absorption. Moreover, the fluorescence lifetimes measured for dinucleotides were shorter than 50 ps, indicating a very fast intramolecular quenching.
The steady-state irradiation of a deoxygenated aqueous solution of a Pyr+-GMP mixture was carried out until approximately 50 % of the substrate was reacted as monitored by HPLC. The photochemical reaction led to the formation of a water insoluble red suspension, which was assigned to the Pyr-Pyr dimer. Other products, particularly those formed by the GMP radical, were not identified during the project. Transient absorption spectra from laser flash photolysis experiments confirmed that Pyr was a primary product of the reaction. Nevertheless, no radicals linked to guanosine (Gua) could be directly observed in flash photolysis experiments.
Lack of the observation of Gua-linked radicals suggested searching for sensitisers that exhibited lower or no absorption in the spectral region in which Gua radicals were expected to absorb. Therefore, the choice was made on 3-Carboxy-benzophenone (3-CB), since its photochemical properties were previously investigated by our group. Based on steady-state and laser flash photolysis results, the draft mechanism of the photosensitised electron transfer reaction was identified and proposed.
Irradiation of a deoxygenated aqueous solution of a Pyr-Thymidine dinucleotide was continued until around 50 % of the substrate was reacted. The photochemical reaction was monitored by HPLC and resulted in the formation of several products. The main product was identified to be a zwitterionic species resulting from a Pyr-base release.
Although deeper insight into the mechanism of the photochemical oxidation processes in more complex DNA systems required further studies, the efforts to understand photo-induced electron transfer reactions in DNA model compounds were successful. As the project would continue beyond its initial timeframe, it was very likely that all the problems suggested in the proposal would be completely solved.
Finally, it should be noted that Tomasz Pedzinski would like to thank Prof. O. Brede and members of his group for their hospitality and assistance in the laser experiments that were performed at the University of Leipzig, Germany.
Moreover, an existing time-resolved fluorescence spectrofluorimeter based on time-correlated single-photon counting detection technique (IBH System 5000) was successfully upgraded. This modification included:
1. exchange of the excitation source to a modern, laser-based diodes (nanoLED);
2. calibration of monochromators;
3. installation and configuration of an analogue to digital (A-D) converter; and
4. software configuration.
The synthesis of the DNA model compounds containing fluorescent N-(purin-6-yl)-pyridinium residues was successfully completed and the products obtained within each synthesis step were controlled using High performance liquid chromatography (HPLC) with Ultraviolet (UV), fluorescence and Mass spectrometry (MS )detection.
Fluorescence quenching experiments were carried out using steady-state and time resolved techniques for intermolecular quenching of highly fluorescent N-(purin-6-yl)-pyridinium chloride by four DNA nucleoside monophosphates. The dynamic plots from the lifetimes were linear and their slopes were practically independent of the quencher used, while the static ones, from the fluorescence intensities, showed significant upward curvature. The latter observation was indicative of a substantial contribution of a static quenching to the overall quenching process; however, no ground state complexation could be observed when studied by UV absorption. Moreover, the fluorescence lifetimes measured for dinucleotides were shorter than 50 ps, indicating a very fast intramolecular quenching.
The steady-state irradiation of a deoxygenated aqueous solution of a Pyr+-GMP mixture was carried out until approximately 50 % of the substrate was reacted as monitored by HPLC. The photochemical reaction led to the formation of a water insoluble red suspension, which was assigned to the Pyr-Pyr dimer. Other products, particularly those formed by the GMP radical, were not identified during the project. Transient absorption spectra from laser flash photolysis experiments confirmed that Pyr was a primary product of the reaction. Nevertheless, no radicals linked to guanosine (Gua) could be directly observed in flash photolysis experiments.
Lack of the observation of Gua-linked radicals suggested searching for sensitisers that exhibited lower or no absorption in the spectral region in which Gua radicals were expected to absorb. Therefore, the choice was made on 3-Carboxy-benzophenone (3-CB), since its photochemical properties were previously investigated by our group. Based on steady-state and laser flash photolysis results, the draft mechanism of the photosensitised electron transfer reaction was identified and proposed.
Irradiation of a deoxygenated aqueous solution of a Pyr-Thymidine dinucleotide was continued until around 50 % of the substrate was reacted. The photochemical reaction was monitored by HPLC and resulted in the formation of several products. The main product was identified to be a zwitterionic species resulting from a Pyr-base release.
Although deeper insight into the mechanism of the photochemical oxidation processes in more complex DNA systems required further studies, the efforts to understand photo-induced electron transfer reactions in DNA model compounds were successful. As the project would continue beyond its initial timeframe, it was very likely that all the problems suggested in the proposal would be completely solved.
Finally, it should be noted that Tomasz Pedzinski would like to thank Prof. O. Brede and members of his group for their hospitality and assistance in the laser experiments that were performed at the University of Leipzig, Germany.