NMR-BASED MODEL OF A TELOMERASE-INHIBITING COMPOUND BOUND TO G-QUADRUPLEX DNA

The single-stranded (TTAGGG)(n) tail of human telomeric DNA is known t o form stable G-quadruplex structures. Optimal telomerase activity req uires the nonfolded single-stranded form of the primer, and stabilizat ion of the G-quadruplex form is known to interfere with telomerase bin ding. We have identified 3,4,9,10-perylenetetracarboxylic diimide-base d ligands as potent inhibitors of human telomerase by using a primer e xtension assay that does not use PCR-based amplification of the telome rase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides relate d to human telomeric DNA showed strong and specific binding of the lig and to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determi nation of the binding site and mode of binding. In the case of the 5'- TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes o riented in a tail-to-tail manner with the ligand sandwiched between te rminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGG TTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligan d bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telom eric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.