Purdue University, College of Pharmacy, Medicinal Chemistry and Molecular Pharmacology, 575 W Stadium Ave, West Lafayette, IN 47907, USA.
University of Arizona, College of Pharmacy, 1703 East Mabel Street, Tucson, AZ 85721, USA.
Biochim Biophys Acta Gen Subj. 2018 Apr;1862(4):846-854. doi: 10.1016/j.bbagen.2017.12.011. Epub 2017 Dec 28.
While the most stable G-quadruplex formed in the human PDGFR-β promoter nuclease hypersensitive element (NHE) is the 5'-mid G-quadruplex, the 3'-end sequence that contains a 3'-GGA run forms a less stable G-quadruplex. Recently, the 3'-end G-quadruplex was found to be a transcriptional repressor and can be selectively targeted by a small molecule for PDGFR-β downregulation.
We use 1D and 2D high-field NMR, in combination with Dimethylsulfate Footprinting, Circular Dichroism Spectroscopy, and Electrophoretic Mobility Shift Assay.
We determine that the PDGFR-β extended 3'-end NHE sequence forms two novel end-insertion intramolecular G-quadruplexes that co-exist in equilibrium under physiological salt conditions. One G-quadruplex has a 3'-non-adjacent flanking guanine inserted into the 3'-external tetrad (3'-insertion-G4), and another has a 5'-non-adjacent flanking guanine inserted into the 5'-external tetrad (5'-insertion-G4). The two guanines in the GGA-run move up or down within the G-quadruplex to accommodate the inserted guanine. Each end-insertion G-quadruplex has a low thermal stability as compared to the 5'-mid G-quadruplex, but the selective stabilization of GSA1129 shifts the equilibrium toward the 3'-end G-quadruplex in the PDGFR-β NHE.
An equilibrium mixture of two unique end-insertion intramolecular G-quadruplexes forms in the PDGFR-β NHE 3'-end sequence that contains a GGA-run and non-adjacent guanines in both the 3'- and 5'- flanking segments; the novel end-insertion structures of the 3'-end G-quadruplex are selectively stabilized by GSA1129.
We show for the first time that an equilibrium mixture of two unusual end-insertion G-quadruplexes forms in a native promoter sequence and appears to be the molecular recognition for PDGFR-β downregulation.
虽然人类 PDGFR-β 启动子核酸酶超敏元件（NHE）中形成的最稳定的 G-四链体是 5'-中端 G-四链体，但包含 3'-GGA 序列的 3'-端序列形成不太稳定的 G-四链体。最近，发现 3'-端 G-四链体是转录抑制剂，并且可以被小分子选择性靶向以降低 PDGFR-β。
我们使用 1D 和 2D 高场 NMR，结合二甲硫酸盐足迹、圆二色性光谱和电泳迁移率变动分析。
我们确定 PDGFR-β 扩展的 3'-端 NHE 序列形成两种新的末端插入的分子内 G-四链体，在生理盐条件下以平衡状态共存。一种 G-四链体具有插入到 3'-外部四联体（3'-插入-G4）的 3'-非相邻侧翼鸟嘌呤，另一种具有插入到 5'-外部四联体的 5'-非相邻侧翼鸟嘌呤（5'-插入-G4）。GGA 序列中的两个鸟嘌呤在 G-四链体内上下移动以容纳插入的鸟嘌呤。与 5'-中端 G-四链体相比，每个末端插入的 G-四链体的热稳定性都较低，但 GSA1129 的选择性稳定化作用使 PDGFR-β NHE 中的 3'-端 G-四链体平衡向有利于 G-四链体的方向移动。
在包含 GGA 序列和 3'-和 5'-侧翼片段中的非相邻鸟嘌呤的 PDGFR-β NHE 3'-端序列中形成平衡混合物的两种独特的末端插入的分子内 G-四链体；新型的 3'-端 G-四链体的末端插入结构被 GSA1129 选择性稳定。
我们首次表明，在天然启动子序列中形成平衡混合物的两种异常的末端插入 G-四链体，并且似乎是 PDGFR-β 下调的分子识别。
