Photochemistry and Photobiology, 1999, zyxwvut 70(6): 829-840 Base-specific Photocleavage of DNA Induced by Pazelliptine Sensitization: Study of the Mechanism by Time-resolved Absorption and Fluorescence Marie-Pierre Fontaine-Aupart*l, Eric Renaultl, Christine Videlot’, Francis Tflbell, Robert Pansu2, Michel Charlier3 and Pascal Pernot4 Laboratoire de Photophysique Moleculaire, Universite Paris-Sud, Orsay, France; 2Laboratoire de Photophysique et Photochimie Supramoleculaires et Macromoleculaires, E.N.S Cachan, Cachan, France; 3Centrede Biophysique Moleculaire, Orleans, France and 4Laboratoirede Physico-Chimie des Rayonnements, Orsay, France Received 13 April 1999; accepted 26 August 1999 ABSTRACT zyxwvutsrq The intercalating antitumoral drug pazelliptine zyxwvu (PZE) is able to photosensitize the formation of single- and dou- ble-strand breaks in supercoiled plasmid DNA and selec- tive photocleavage at guanine residues is observed. In or- der to understand the mechanisms of DNA cleavage me- diated by the photoexcited drug, singlet and triplet ex- cited-state processes in PZE complexed with poly(dA- dT)-poly(dA-dT), poly(dG-dC).poly(dG-dC) and calf thymus DNA have been investigated by means of single photon counting fluorescence decay and transient ab- sorption techniques. For each complex, three different binding sites have been identified, due to the existence of different geometric structures of the drug in the ground state. For one type of binding site, a proton transfer re- action occurs in the singlet excited state whatever the nucleic acid environment. In contrast, the relaxation dy- namics for the other two sites are found to depend widely upon the type of polynucleotide in which the drug has been intercalated. From the results of this study, we sug- gest that the photodynamic action of PZE does not orig- inate from excitation of the drug in the environment of G-C base pairs but is initiated from its triplet state that reacts by electron transfer with the adenine bases. The specificity of cleavage could be the result of subsequent reactions leading to guanine oxidation. INTRODUCTION There is a growing interest in the research of classes of mol- ecules able to act both as drug and nano-tools for detecting and manipulating the genetic material. Many active antitu- moral agents of the DNA-intercalating category, possessing sequence-specific binding sites have already been proved to be useful as sensitive probes of local nucleic acid sequence and topology, as well as site-specific reagents for molecular biology (DNA sequencing, antigen strategy). The motivation for these studies increases if the DNA-targeted molecules are also photosensitizers of the nucleic acid cleavages, be- cause light can be used then as a trigger of the nuclease activity. Ellipticine and its analogues constitute an important class of antitumoral molecules that has been widely studied in terms of their binding to DNA and the mechanisms involved in their biological activity (1-4). Indeed, the DNA damage observed, such as irreversible strand breaks (5-7), is not only the consequence of structural modifications related to the binding of the drug but also of different functional effects such as drug redox reactions (5). Evidence for photosensi- tizing properties of these molecules has been reported in only one specific case (8). In the last few years, we have focused our attention on pazelliptine (PZE),t one of the most active derivatives of ellipticine (Fig. 1). We have reported its strong interaction with DNA zyxw (K,,, - zy lo6 zyxw M-I) and confirmed that the drug is intercalated into DNA by fluorescence energy transfer ex- periments (9). The possibility of a photosensitizing effect of the drug toward DNA was suggested, considering the effi- cient fluorescence quenching (80-90%) observed when PZE was complexed to calf thymus DNA and poly(dG- dC).poly(dG+IC), a process frequently observed with pho- tosensitizers (10-12). In the present paper, we have first proved the ability of PZE to photosensitize DNA cleavage and looked for the preferential location of cleavage by sequencing gel electro- phoresis. In a second step, we have tried to elucidate the possible mechanism(s) for its photodynamic activity using time-resolved absorption and fluorescence spectroscopy. A preliminary subpicosecond transient absorption study re- vealed the activation of a radiationless transition of the sin- *To whom correspondence should be addressed at: Laboratoire de Photophysique Moleculaire, U.P.R. 3361 CNRS, Batiment 213, Universitt Paris-Sud, 91405 Orsay Cedex, France. Fax: 01 69 15 67 77; e-mail: marie-pierre.fontaine-aupart C3ppm.u-psud.fr zyxwvuts 0 1999 American Society for Photobiology 0031-8655/99 $5.00+0.00 tAbbreviations: bp, base pairs; CD, circular dichroism; CT-DNA, calf thymus DNA; DSB, double-strand breaks; PZE, pazelliptine; SAS, species-associated spectra; SSB, single-strand breaks; SVD, singular value decomposition.