Tandem triplex formation on the promoter of the c-myc gene with daunomycin-conjugated oligonucleotides

It is postulated that a control of cancer cell proliferation would be possible through an interference with the expression of relevant oncogenes (e. g. c-myc) via formation of a triple helix (triplex) on a selected region of the oncogene. TFO-daunomycin conjugates (D-TFOs) have been proved to inhibit the expression of a gene controlled by the c-myc promoter (Catapano et al. Abstract #4563 AACR 2000). D-TFOs are endowed with the ability to bind selectively to the target region of the c-myc promoter through the formation of Hoogsteen's type hydrogen bonds, the stability of the resulting complex being enhanced by (1) the intercalation of the anthraquinone planar system between adjacent base pairs of the DNA and (2) the favorable interaction of the aminosugar moiety in the minor groove. Together with a significant improvement of the synthetic process further progress has been made in the direction of providing more effective binders. Synthesis of conjugates has been performed using new protecting intermediates followed by appropriate deblocking of the final products. Studies of the stability of complexes is carried out using PAGE mobilitiy shift experiments. Linkage of daunomycin to both ends of TFO rsults in conjugates that bind to the appropriate DNA sequence with enhanced stability corresponding to asociation constants in the micromolar range at 37 °C and pH 7.0 as determined using hybridization on a model duplex. Additionally, a new interesting approach is bsesed on the formation of two different triplex on contiguous segments of the targeted region (tandem triplex formation). The results show an enhancement of the stabilization of about one order of magnitude as compared with the single triplex complex. In conclusion D-TFOs appear to be promising candidates for the development of gene expression inhibitors.