Anthracycline G-Quadruplex DNA complexes: circular dichroism, fluorescence and thermodynamic features

Anthracyclines are a class of antitumoral drugs widely used in clinical practice. Even though it is generally accepted that they target DNA, it is still not clear how anthracycline drugs exactly work. While a wealth of data are available on the interaction of anthracyclines with duplex DNA, little is known about the binding to other forms of DNA, like the G-quadruplex (G4). G4 structures are secondary DNA structures adopted by guanine-rich DNA sequences and consist of - stacked planes of four guanosines cyclically bound to each other via 8 hydrogen bonds according to the Hoogsteen motif. The structure is stabilized by the presence of monovalent cations like Na+ and K+. Human telomeres at the end of the chromosomes consist of thousands of repeats of the guanine-rich sequence 5’-d(GGGTTA)-3’ and possess at the 3’ end a single-stranded overhang of 100-200 bases mainly consisting of the same sequence. Telomere shortening during cell replication is one of the factors accounting for cell senescence. A telomere maintenance mechanism is mediated by the telomerase protein complex. Overexpression of this protein has been observed for rapidly replicating tumor cells guaranteeing their “immortality”. The G-quadruplex structure seems to play a significant role in this biological process. Moreover guanine-rich sequences are present in promoter regions of many oncogenes and compelling evidence exists that they assume a G4 structure in solution. Consequently guanine-rich sequences of both telomeres and oncogenes have attracted a lot of research interest during the last decade as target for antitumoral drug action.1 Using an approach that combines fluorescence, absorption, circular dichroism and microcalorimetric techniques we proved that Doxorubicin, also called Adriamycin, and Sabarubicin, also known as MEN 10755, strongly bind to a model human telomeric sequence, the 21mer 5’-d[GGG(TTAGGG)3]-3’, assuming G4 structure. Both 1:1 and 2:1 drug:21mer complexes coexist in solution. Association constants (~ 105 -106 M-1) were obtained from fluorescence titration experiments and were confirmed by isothermal titration calorimetry (ITC). The spectroscopic features of the individual complexes were extracted from the spectra of the mixtures. The circular dichroism in the UV region indicates that drug binding influences the equilibrium between various G-quadruplex conformations, whereas in the visible region it gives information about the drug complexation modes. The presence of either K+ or Na+ controls the G4 conformation and reflects in the spectroscopic properties of the complexes. Thermodynamic data suggested that binding involves stacking on top/bottom G-tetrad and insertion into grooves. Complexation causes a small increase of the thermal stability of the G4 conformation in the presence of K+, while it does not affect the thermal stability of the G4 structure in the presence of Na+.