【作者】 李春燕；

【导师】 任艾明；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2024， 博士

【摘要】 核开关和RNA适配体是两类可以特异结合配体的非编码RNA分子。核开关是一类天然存在的RNA元件,主要位于细菌m RNA的5’UTR区域,通过识别特定的代谢物小分子传递构象变化,对下游基因的表达起着重要的调控作用。RNA适配体是一类通过体外筛选（SELEX）技术获得的具有特定结构的RNA,可以特异性地结合配体如小分子等,在细胞成像、疾病诊断和基因治疗中具有广泛的应用潜力。四氢叶酸（THF）Ⅱ型核开关是近期鉴定的第二类识别THF的新型核开关,其参与调控革兰氏阴性菌内THF的代谢稳态。环丙沙星（CFX）适配体是经过体外SELEX和体内筛选共同鉴定的RNA适配体,是首类可以结合氟喹诺酮类抗生素的RNA适配体,研究发现,CFX适配体还可以在细胞内发挥核开关调控下游基因表达的功能。然而,上述两类RNA分子的三维结构、各自识别配体的作用方式以及它们的调控机制均不清楚,本论文采用X-射线晶体学的方法分别解析了THF-Ⅱ型核开关和CFX适配体的三维结构,阐述了两类RNA识别相应配体的分子机制。本论文的第一个课题解析了THF-Ⅱ型核开关-THF复合物的晶体结构,阐明了THF-Ⅱ型核开关的配体识别与基因调控机制。THF-Ⅱ型核开关整体呈现杆状结构,J12区域内部形成复杂的氢键网络,L2环与J12的碱基堆积作用维持了J12区域的折叠,这些远程相互作用共同促进了结合口袋的稳定。结构分析表明,该核开关通过三碱基作用层面识别THF的四氢蝶呤部分,并将THF配体包裹在J12与stem P2形成的结合腔中。此外,本研究还解析了THF-Ⅱ型核开关free-form的结构,并通过CD和ITC等实验手段阐明了镁离子对于THF-Ⅱ型核开关折叠的重要性。基于这些结构信息,本研究还鉴定了与THF-Ⅱ型核开关相互作用的其他化合物,包括天然代谢产物（DHF、BH₄、DHN和Guanine）和潜在的药物化合物（8-N Guanine、8-NH₂Guanine和8-CH₃Guanine）。综上所述,对于THF-Ⅱ型核开关三维结构的研究不仅阐明了其识别配体的分子机制,为生命体维持THF代谢稳态的机制提供了更多的研究基础,同时也为开发靶向THF-Ⅱ型核开关的抗生素药物提供了必要的结构信息。本论文的第二个课题解析了CFX适配体-CFX复合物的三维结构,研究了CFX适配体的作用机制。该结构整体呈现“n”形,stem P1、stem P2和假结PS形成同轴螺旋,假结PS的形成使得stem P3内部的核苷酸呈现出复杂的远程相互作用,并促进配体结合口袋的折叠。该适配体通过碱基堆积作用以及镁离子介导的氢键网络将CFX包裹在stem P3底部区域形成的空腔内。基于对该三维结构的分析,我们优化并获得了可以结合CFX的最小适配体模块P1U4。此外,本研究还解析了CFX适配体与其他三种氟喹诺酮类抗生素（DFX、EFX和NFX）的三维结构。综上所述,对CFX适配体三维结构的研究阐明了其识别氟喹诺酮类抗生素的分子机制,为开发生物传感器和适体酶等工具提供了结构基础。更多还原

【Abstract】 Riboswitches and RNA aptamers are two types of non-coding RNA molecules that can bind to their cognate ligands.Riboswitches are naturally occurring RNA elements primarily located in the 5’UTR of bacterial m RNA.They play an important regulatory role in downstream gene expression by recognizing specific metabolite molecules to transmit conformational changes.RNA aptamers are a type of RNA with specific structures selected in vitro by SELEX（Systematic Evolution of Ligands by Exponential Enrichment）technology.They can specifically bind to small molecules and have broad potential application in cell imaging,disease diagnosis,and gene therapy.Tetrahydrofolate（THF）-Ⅱ riboswitch is a recently identified second class of riboswitch that can bind to THF and is involved in regulating the intracellular levels of THF in Gram negative bacteria.Ciprofloxacin（CFX）aptamer is an RNA aptamer identified through in vitro SELEX and in vivo screening.It is the first class of RNA aptamers that can bind to fluoroquinolone antibiotics and function as riboswitch in cells,regulating the expression of downstream gene.However,the tertiary structure of these two types of RNA molecules,the way they recognize their respective ligands,and their regulatory mechanisms are still unclear.In this theory,we used X-ray crystallography to solve the three-dimensional structures of THF-Ⅱ riboswitch and CFX aptamer,therefore elucidating the underlying molecular mechanism of ligand recognition.In the first project,we determined the crystal structure of THF-Ⅱ riboswitch-THF complexes and identified the ligand recognition and gene regulation mechanism of THF-Ⅱ riboswitch.Overall,The THF-Ⅱ riboswitch exhibits a rod-like conformation.A complex hydrogen-bond network is formed within the junction J12,and the stacking interaction between the L2 loop and junction J12 maintains the folding of the J12 region.These long-distance interactions together promote the stability of the binding pocket.Structural analysis indicates that the THF-Ⅱ riboswitch recognizes the tetrahydropterin moiety of THF through the triple base interaction and clamping the ligand within the binding cavity formed by junction J12 and stem P2.In addition,we also solved the free-form structure of THF-Ⅱ riboswitch and demonstrated the importance of magnesium ions in THF-Ⅱ riboswitch folding through CD and ITC experiments.Notably,we also identified other compounds that interact with THF-Ⅱ riboswitch,including natural metabolites（DHF,BH₄,DHN,and Guanine）and potential drug compounds（8-N Guanine,8-NH₂Guanine,and 8-CH₃Guanine）.In summary,the study of the three-dimensional structure of THF-Ⅱ riboswitch not only elucidates the molecular mechanism of ligand recognition,but also provides more insights into the mechanism of maintaining THF metabolic level in organisms.In the second project,we focused on the crystal structure of CFX aptamer-CFX complexes.Overall,The CFX aptamer adopts a“n-shape”conformation,in which the two stems P1,P2 and pseudoknot PS form coaxial helices.The formation of pseudoknot PS leads to complex long-distance interactions between nucleotides within stem P3 and promotes the folding of ligand binding pocket.This aptamer encapsulates CFX in the binding cavity formed by the bottom region of stem P3through base stacking and hydrogen network mediated by magnesium ions.Based on the structural analysis,we optimized the sequence of CFX aptamer and obtained the minimum RNA motif P1U4 that can recognize CFX ligand.Moreover,we also solved the three-dimensional structure of the CFX aptamer and three other fluoroquinolone antibiotics（DFX,EFX and NFX）.In summary,the study of the three-dimensional structure of CFX aptamer has elucidated their recognition mechanism for fluoroquinolone antibiotics and provided a structural basis for the development of biosensors,aptazymes and other tools.更多还原