title: Stabilization of G-quadruplex in the BCL2 promoter region in double-stranded DNA by invading short PNAs.
abstract: Numerous regulatory genes have G-rich regions that can potentially form quadruplex structures, possibly playing a role in transcription regulation. We studied a G-rich sequence in the BCL2 gene 176-bp upstream of the P1 promoter for G-quadruplex formation. Using circular dichroism (CD), thermal denaturation and dimethyl sulfate (DMS) footprinting, we found that a single-stranded oligonucleotide with the sequence of the BCL2 G-rich region forms a potassium-stabilized G-quadruplex. To study G-quadruplex formation in double-stranded DNA, the G-rich sequence of the BCL2 gene was inserted into plasmid DNA. We found that a G-quadruplex did not form in the insert at physiological conditions. To induce G-quadruplex formation, we used short peptide nucleic acids (PNAs) that bind to the complementary C-rich strand. We examined both short duplex-forming PNAs, complementary to the central part of the BCL2 gene, and triplex-forming bis-PNAs, complementary to sequences adjacent to the G-rich BCL2 region. Using a DMS protection assay, we demonstrated G-quadruplex formation within the G-rich sequence from the promoter region of the human BCL2 gene in plasmid DNA. Our results show that molecules binding the complementary C-strand facilitate G-quadruplex formation and introduce a new mode of PNA-mediated sequence-specific targeting.
authors.affiliation: Department of Radiology and Imaging Sciences, Clinical, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Mykola I
authors.initials: MI
authors.lastname: Onyshchenko
authors.forename: Timur I
authors.initials: TI
authors.lastname: Gaynutdinov
authors.forename: Ethan A
authors.initials: EA
authors.lastname: Englund
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Appella
authors.forename: Ronald D
authors.initials: RD
authors.lastname: Neumann
authors.forename: Igor G
authors.initials: IG
authors.lastname: Panyutin
mesh_terms: D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D018983:DNA Footprinting; D054856:G-Quadruplexes; D019254:Genes, bcl-2; D006801:Humans; D009691:Nucleic Acid Denaturation; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry; D011401:Promoter Regions, Genetic
chemical_list: D020135:Peptide Nucleic Acids; D004247:DNA