Nucleic Acids Research, 2000, Vol. 28, No. 12 2307-2310
© 2000 Oxford University Press
The role of polyamines, Na+ and K+ in the formation of triple helices between purine oligonucleotides and the promoter region of the human c-src proto-oncogene
Department of Biochemistry, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada and 1Department of Medicine, Robert Wood Johnson Medical School, 125 Paterson Street, New Brunswick, NJ 08903, USA
Binding constants for triplex formation between purine-rich oligonucleotides and a pyrimidine·purine tract of the human c-src proto-oncogene were measured by fluorescence polarization in the presence of polyamines, Na+ and K+. In both the hexamine and tetramine series, the longer polyamines had the larger binding constants for triplex formation at low concentrations of polyamine. At higher concentrations all values tended to plateau in the 109/M range. In contrast to previous reports, K+ did not inhibit triplex formation and at 150 mM the binding constants were again in the 109/M range for both an 11mer and 22mer oligonucleotide. At 150 mM K+ the addition of polyamines did not lead to any significant increase in the binding constants. It was determined that the lack of inhibition by K+ was due to the low concentration (1 nM) of purine oligonucleotide required for the fluorescence polarization technique. At higher concentrations (1 µM) self-association of the oligonucleotide was observed. These results suggest that in vivo, at least for the c-src promoter, the inhibition of triplex formation by K+ may not be detrimental. However, it may be difficult to achieve binding constants above ~109/M even in the presence of polycations.
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