Antiproliferative activity of G-quartet-forming oligonucleotides with backbone and sugar modifications

Abstract

Oligonucleotide-based therapies have considerable potential in cancer, viral, and cardiovascular disease therapies. However, it is becoming clear that the biological effects of oligonucleotides are not solely due to the intended sequence-specific interactions with nucleic acids. Oligonucleotides are also capable of interacting with numerous cellular proteins owing to their polyanionic character or specific secondary structure. We have examined the antiproliferative activity, protein binding, and G-quartet formation of a series of guanosine-rich oligonucleotides, which are analogues of GRO29A, a G-quartet forming, growth-inhibitory oligonucleotide, whose effects we have previously described [Bates P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377]. The GRO29A analogues include phosphorothioate (PS29A), 2′-O-methyl RNA (MR29A), and mixed DNA/ 2′-O-methyl RNA (MRdG29A) oligonucleotides. We demonstrate by UV spectroscopy that all of the modified analogues form stable structures, which are consistent with G-quartet formation. We find that the phosphorothioate and mixed DNA/2′-O-methyl analogues are able to significantly inhibit proliferation in a number of tumor cell lines, while the 2′-O-methyl RNA has no significant effects. Similar to the original oligonucleotide, GRO29A, the growth inhibitory oligonucleotides were able to compete with the human telomere sequence oligonucleotide for binding to a specific cellular protein. The less active MR29A does not compete significantly for this protein. On the basis of molecular modeling of the oligonucleotide structures, it is likely that the inactivity of MR29A is due to the differences in the groove structure of the quadruplex formed by this oligonucleotide. Interestingly, all GRO29A analogues, including an unmodified DNA phosphodiester oligonucleotide, are remarkably resistant to nuclease degradation in the presence of serum-containing medium, indicating that secondary structure plays an important role in biological stability. The remarkable stability and strong antiproliferative activity of these oligonucleotides confirm their potential as therapeutic agents.