Poster Sessions

SB -10

Mykola Onyshchenko

M. Onyshchenko, R. D. Neumann, I. Panyutin

G-quadruplex stabilization in double-stranded DNA by short PNA designed to bind to complementary strand

G-quadruplexes as secondary structures in DNA are the object of extremely intensive research nowadays. There is accumulating in vitro evidence of their formation in G-rich regulatory sequences of several genes, including promoter regions, where quadruplexes can play a role of transcriptional regulators working as anchors for transcriptional proteins. These genes include, but not limited to c-myc, bcl2, transcriptional enhancer reported in the human insulin gene, and several muscle-specific genes. In our study we showed the possibility of developing the conditions where in particular regions of double-stranded plasmid DNA quadruplexes can be formed due to binding to the complementary strand of short PNA. pCRBlunt 3.5 Kb plasmid DNA was used as a model with following inserts: G-rich region of bcl2 gene and four GGGGTT repeats from Tetrahymena thermophila telomere. Besides, plasmid samples with various values of superhelicity with sigma starting from 0 to -0.13 have been studied. Chemical and enzymatic probings were used as a technique for detecting open regions of DNA; DMS footprinting was used for quadruplex detection by N7 position protection in guanines. Clear evidence of quadruplex formation in studied inserts as oligos under different incubation was obtained. The results proved the possibility of stable complementary binding of short (starting from 6 bases) PNAs to double-stranded naturally supercoiled DNA at overnight incubation at 37C with simultaneous opening of the second G-rich strand. After getting PNA bound, various following incubation conditions, including potassium, sodium, TMPyP4 and Telomestatin, are studied to obtain clear evidence of quadruplex formation.