22-nt端粒RNA片段所形成反向平行G-四链体的构型影响要素分析

Analysis of the Factors Affecting the Configuration of the Anti-parallel G-quadruplex Formed by 22-nt Telomere RNA Fragments

不同构象的G-四链体通常展现出与蛋白质结合能力不同,进而发挥出不同的生理功能,分析G-四链体的构型影响要素,对探究其生理功能有重要意义。不同于DNA,RNA序列在以往的研究中均呈现平行G-四链体结构,前期研究中我们发现并报道了目前唯一能形成反向平行G-四链体的RNA序列,22-nt端粒RNA片段(ORN-N,A_(1)G_(2)G_(3)G_(4)U_(5)U_(6)A_(7)G_(8)G_(9)G_(10)U_(11)U_(12)A_(13)G_(14)G_(15)G_(16)U_(17)U_(18)A_(19)G_(20)G_(21)G_(22))。分析ORN-N的构型影响因素将极大拓展对RNA特性的认识。因此,本研究旨在系统性的探索能够影响ORN-N构型的因素。使用圆二色谱和熔融曲线监测糖环2′-羟基对ORN-N构型和稳定性的影响,发现ORN-N中不同核苷糖环2′-羟基对于反向平行G-四链体的作用不同。U_(6)、A_(7)、G_(16)的糖环2′-羟基对ORN-N的拓扑构象-维持至关重要,G_(3)、A_(13)、A_(19)的糖环2′-羟基具有稳定其构象的作用,而U_(17)的糖环2′-羟基降低了ORN-N的稳定性,此与以往研究中糖环2′-羟基不利于RNA序列形成反向平行G-四链体构型不同。将ORN-N环中的核苷U进行5-甲基化修饰,通过圆二色谱和熔融曲线监测其构象和稳定性,发现环中部分U碱基5-甲基化能稳定ORN-N的构象。使用5%~40%PEG模拟细胞内分子拥挤环境,通过圆二色谱监测其构象,结果与以往研究不同,分子拥挤环境并不能稳定反向平行RNA G-四链体,相反ORN-N的构象发生了翻转。综上,在ORN-N中糖环2′-羟基、环序列甲基化以及分子拥挤环境对反向平行RNA G-四链体的构型具有极其重要的影响。

G-quadruplexes with different conformations often exhibit different binding abilities to proteins and play different physiological functions.It is of great significance to analyze the factors affecting the configuration of G-quadruplex to explore its physiological function.Different from DNA,RNA sequences have shown parallel G-quadruplex structures in previous reports.However,we found that the only RNA sequence,the 22-nt telomere RNA fragment(ORN-N,A_(1)G_(2)G_(3)G_(4)U_(5)U_(6)A_(7)G_(8)G_(9)G_(10)U_(11)U_(12)A_(13)G_(14)G_(15)G_(16)U_(17)U_(18)A_(19)G_(20)G_(21)G_(22)),can form the anti-parallel G-quadruplex in the previous study.Analyzing the fac-tors affecting the configuration of ORN-N will greatly expand the understanding of the characteristics of RNA.Therefore,the purpose of this study is to systematically explore the factors that can affect ORN-N configuration.The effects of the 2′hydroxyl of the ribose sugar on the configuration and stability of ORN-N were detected by circular dichroism and melting curve.We found that the 2′hydroxyl of the ribose sug-ar of different nucleosides in ORN-N had different effects on the anti-parallel G-quadruplex.For exam-ple,the 2′hydroxyl of the ribose sugar of U_(6),A_(7),and G_(16)is crucial to the maintenance of the topological conformation of ORN-N.The 2′hydroxyl of the ribose sugar of G_(3),A_(13),and A_(19)can stabilize its confor-mation,while the 2′hydroxyl of the ribose sugar of U_(17)reduces the stability of ORN-N.This is different from the previous studies that the 2′hydroxyl of the ribose sugar is not conducive to the formation of anti-parallel G-quadruplex structure of RNA.After 5-methylating the U base in the loop of ORN-N,its confor-mation and stability were detected by circular dichroism and melting curve.We found that some U base 5-methylation in the loop can stabilize the conformation of ORN-N.We simulated an intracellular molecu-lar crowding environment using 5%~40%PEG,and then monitored the conformation of ORN-N through circular dichroism.Different from previous studies,the molecular crowding environment can not stabilize the anti-parallel RNA G-quadruplex.On the contrary,the conformation of ORN-N has been reversed.In summary,the 2′hydroxyl of the ribose sugar,loop sequence methylation,and the molecular crowding environment have extremely important effects on the configuration of the anti-parallel RNA G-quadruplex in ORN-N.