Department of Chemistry, Mississippi State University, Mississippi State, Mississippi, USA.
Biophys J. 2011 Mar 16;100(6):1517-25. doi: 10.1016/j.bpj.2011.01.068.
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).
我们使用差示扫描量热法、等温滴定量热法和圆二色光谱法完成了 c-MYC 原癌基因 P1 启动子四联体及其与阳离子卟啉 5,10,15,20-四（N-甲基-4-吡啶基）卟啉（TMPyP4）相互作用的生物物理特性分析。我们研究了三种不同的 24 -mer 寡核苷酸，包括 c-MYC P(1)启动子中发现的野生型（WT）序列和两种已知折叠成单 1:2:1 和 1:6:1 环式异构体四联体的突变 G→T 序列。在两种不同离子强度（30 mM [K(+)]和 130 mM [K(+)]）下，对所有三种寡核苷酸序列进行了生物物理实验。差示扫描量热法实验表明，WT 四联体在两种离子强度下都由至少两种不同折叠构象的混合物组成，而两种突变序列在两种离子强度下都表现出单一的两态熔解转变。等温滴定量热法实验表明，两种突变序列都能与 1 摩尔 DNA 结合 4 摩尔 TMPyP4，与 WT 序列相似。在两种离子强度下，所有三种寡核苷酸的圆二色光谱特征都与分子内平行链 G-四联体结构一致，并且在加入饱和量的 TMPyP4（即 4:1 TMPyP4/DNA）时，四联体结构没有变化。
