Kumar Niti, Maiti Souvik
Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR Mall Road, New Delhi 110 007, India.
Nucleic Acids Res. 2005 Nov 30;33(21):6723-32. doi: 10.1093/nar/gki961. Print 2005.
The structural competition between the G-quadruplex and Watson-Crick duplex has been implicated for the repetitive DNA sequences, but the factors influencing this competitive equilibrium in the natural and pharmacological context need to be elucidated. Using a 21mer 5'-Fluorescein-d[(G3TTA)3G3]-TAMRA-3' as a model system, extensive fluorescence resonance energy transfer analysis was carried out to investigate sensitivity of this equilibrium to osmotic stress and quadruplex selective small molecule. The binding affinities and kinetics involved in the hybridization of quadruplex to its complementary strand in the absence and presence of different concentrations of osmolytes (ethylene glycol and glycerol) and a quadruplex selective ligand (cationic porphyrin-TMPyP4) were determined. The presence of osmolytes and cationic porphyrin decreased the binding affinity of quadruplex to its complementary strand and slowed the kinetics of the reaction by delaying the hybridization process. Our binding data analysis indicates that the presence of either osmolytes or porphyrin increase the amount of quadruplex in the equilibrium. In 100 mM KCl solution, when 30 nM of each of the components, i.e. quadruplex and the complementary strand, were mixed together, the amount of quadruplex present in the system under equilibrium were 17.6, 23.4, 23.1 and 19.6 nM in the absence and presence of 10% ethylene glycol, 10% glycerol and 150 nM TMPyP4, respectively. Fluorescence melting profile of quadruplex in the absence and presence of these perturbants confirm the findings that osmolytes and cationic porphyrin stabilize quadruplex, and thus, shift the equilibrium to quadruplex formation.
G-四链体与沃森-克里克双链体之间的结构竞争与重复DNA序列有关，但在自然和药理学背景下影响这种竞争平衡的因素仍有待阐明。使用21聚体5'-荧光素-d[(G3TTA)3G3]-TAMRA-3'作为模型系统，进行了广泛的荧光共振能量转移分析，以研究这种平衡对渗透应激和四链体选择性小分子的敏感性。测定了在不存在和存在不同浓度的渗透剂（乙二醇和甘油）以及四链体选择性配体（阳离子卟啉-TMPyP4）的情况下，四链体与其互补链杂交所涉及的结合亲和力和动力学。渗透剂和阳离子卟啉的存在降低了四链体与其互补链的结合亲和力，并通过延迟杂交过程减缓了反应动力学。我们的结合数据分析表明，渗透剂或卟啉的存在增加了平衡中四链体的数量。在100 mM KCl溶液中，当将30 nM的每种组分（即四链体和互补链）混合在一起时，在不存在和存在10%乙二醇、10%甘油和150 nM TMPyP4的情况下，系统中平衡时存在的四链体数量分别为17.6、23.4、23.1和19.6 nM。在不存在和存在这些干扰剂的情况下，四链体的荧光熔解曲线证实了渗透剂和阳离子卟啉稳定四链体的发现，因此，将平衡转移到四链体形成。
