title: DSC deconvolution of the structural complexity of c-MYC P1 promoter G-quadruplexes.
abstract: We completed a biophysical characterization of the c-MYC proto-oncogene P1 promoter quadruplex and its interaction with a cationic porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4), using differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy. We examined three different 24-mer oligonucleotides, including the wild-type (WT) sequence found in the c-MYC P(1) promoter and two mutant G→T sequences that are known to fold into single 1:2:1 and 1:6:1 loop isomer quadruplexes. Biophysical experiments were performed on all three oligonucleotide sequences at two different ionic strengths (30 mM [K(+)] and 130 mM [K(+)]). Differential scanning calorimetry experiments demonstrated that the WT quadruplex consists of a mixture of at least two different folded conformers at both ionic strengths, whereas both mutant sequences exhibit a single two-state melting transition at both ionic strengths. Isothermal titration calorimetry experiments demonstrated that both mutant sequences bind 4 mols of TMPyP4 to 1 mol of DNA, in similarity to the WT sequence. The circular dichroism spectroscopy signatures for all three oligonucleotides at both ionic strengths are consistent with an intramolecular parallel stranded G-quadruplex structure, and no change in quadruplex structure is observed upon addition of saturating amounts of TMPyP4 (i.e., 4:1 TMPyP4/DNA).
authors.affiliation: Department of Chemistry, Mississippi State University, Mississippi State, Mississippi, USA.
authors.forename: Jamie M
authors.initials: JM
authors.lastname: Dettler
authors.forename: Robert
authors.initials: R
authors.lastname: Buscaglia
authors.forename: Vu H
authors.initials: VH
authors.lastname: Le
authors.forename: Edwin A
authors.initials: EA
authors.lastname: Lewis
mesh_terms: D002152:Calorimetry, Differential Scanning / Q000379:methods*; D054856:G-Quadruplexes; D009154:Mutation; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011166:Porphyrins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D011687:Purines / Q000378:metabolism; D013816:Thermodynamics; D044366:Transition Temperature
chemical_list: D011166:Porphyrins; D016271:Proto-Oncogene Proteins c-myc; D011687:Purines; C021096:tetra(4-N-methylpyridyl)porphine