Department of Chemistry, University of Burgos, 09001 Burgos, Spain.
Department of Chemistry, University of Burgos, 09001 Burgos, Spain; Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Università degli Studi di Palermo, Viale delle Scienze Ed. 17, 90128 Palermo, Italy.
Biochim Biophys Acta Gen Subj. 2018 Mar;1862(3):522-531. doi: 10.1016/j.bbagen.2017.10.020. Epub 2017 Oct 31.
Stabilization of G-quadruplex helices by small ligands has attracted growing attention because they inhibit the activity of the enzyme telomerase, which is overexpressed in >80% cancer cells. TMPyP4, one of the most studied G-quadruplex ligands, is used as a model to show that the ligands can exhibit different binding features with different conformations of a human telomeric specific sequence.
UV-Vis, FRET melting Assay, Isothermal Titration Calorimetry, Time-resolved Fluorescence lifetime, T-Jump and Molecular Dynamics.
TMPyP4 yields two different complexes with two Tel22 telomeric conformations in the presence of Na or K. T-Jump kinetic experiments show that the rates of formation and dissociation of these complexes in the ms time scale differ by one order of magnitude. MD simulations reveal that, in K buffer, "hybrid 1" conformation yields kinetic constants on interaction with TMPyP4 one order lower than "hybrid 2". The binding involves π-π stacking with external loop bases.
For the first time we show that for a particular buffer TMPyP4 interacts in a kinetically different way with the two Tel22 conformations even if the complexes formed are thermodynamically indistinguishable.
G-quadruplexes, endowed with technological applications and potential impact on regulation mechanisms, define a new research field. The possibility of building different conformations from same sequence is a complex issue that confers G-quadruplexes very interesting features. The obtaining of reliable kinetic data constitutes an efficient tool to determine reaction mechanisms between conformations and small molecules.
小分子稳定 G-四链体螺旋已引起越来越多的关注，因为它们可以抑制端粒酶的活性，端粒酶在超过 80%的癌细胞中过度表达。TMPyP4 是研究最多的 G-四链体配体之一，被用作模型来表明配体可以表现出与不同构象的人端粒特异性序列不同的结合特征。
紫外可见光谱、荧光共振能量转移熔解实验、等温滴定微量热法、时间分辨荧光寿命、T-Jump 和分子动力学。
TMPyP4 在存在 Na 或 K 的情况下与两种 Tel22 端粒构象产生两种不同的复合物。T-Jump 动力学实验表明，这些复合物在毫秒时间尺度上的形成和解离速率相差一个数量级。MD 模拟表明，在 K 缓冲液中，“杂交 1”构象与 TMPyP4 相互作用的动力学常数比“杂交 2”低一个数量级。结合涉及与外部环碱基的π-π堆积。
我们首次表明，对于特定的缓冲液，TMPyP4 以动力学上不同的方式与两种 Tel22 构象相互作用，即使形成的复合物在热力学上无法区分。
具有技术应用和对调节机制潜在影响的 G-四链体定义了一个新的研究领域。从相同的序列构建不同构象的可能性是一个复杂的问题，赋予 G-四链体非常有趣的特征。获得可靠的动力学数据是确定构象与小分子之间反应机制的有效工具。
