NMR Studies of RNA Aptamers for Biological CoFactors

生物辅因子 RNA 适体的 NMR 研究

• 批准号: 9506913
• 负责人: Juli Feigon
• 金额: $ 27万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9506913&HistoricalAwards=false
• 关键词: NMR; Studies; RNA; Aptamers; Biological

9506913 Feigon Aptamer is a name coined for DNA or RNA oligonucleotides which have been selected from a large pool of oligonucleotides containing a region of random nucleotide sequence for binding to a specific target molecule. The isolation process involves repeated cycles of selection for, and enrichment of, oligonucleotides with an affinity to a specific target, followed by amplification of these sequences using the polymerase chain reaction (PCR). Olionucleotides with the selected characteristics, i.e. binding to a specific molecule, are finally identified through cloning and sequencing. The cloned sequences are usually screened for a common consensus and possible secondary structural motif. This methodology has been used to identify a number of different classes of RNA aptamers that bind specifically to common metabolic substrates and cofactors. These include ATP, GTP, riboflavin, FAD, FMN, cyanocobalamin and the amino acids arginine and citrulline. Based on the consensus sequences and further experiments such as deletion analysis, minimal binding sequences have in most cases been identified and a secondary structure proposed. However, to date, no three dimensional structure for RNA aptamers have been determined. Multidimensional, multinuclear NMR spectroscopy will be used to determine the three-dimensional structures of aptamers that bind biological cofactors, in the absence and presence of bound cofactor. RNA samples for NMR studies will be synthesized by in vitro transcription using T7 RNA polymerase. These will initially be investigated by 1H NMR spectroscopy to screen for sequences which give good NMR spectra. Once well-behaved samples are obtained, partially or uniformly 15N and/or 13C-labeled samples will be synthesized, to facilitate complete resonances assignment. In favorable cases, complete three-dimensional structures will be determined using metric matrix distance geometry and refinement by molecular dynamics. %%% NMR spectroscopy will be used to study the structures of RNA oligonucleotides that bind biological cofactors. These RNAs contain a wide variety of proposed secondary structural motifs, including hairpin loops, internal loops, pseudoknots, and intramolecular G-quartets. Thus, determination of their structures will lead to a greater understanding of the principles of nucleic acid folding. Very few RNA structures of any kind have been solved so far, so any information on tertiary structures of RNA should greatly add to the knowledge base of these secondary structure motifs. Determination of the specific cofactor-RNA interactions will also help elucidate some of the factors governing sequence specific recognition of RNA by proteins and other ligands. The structures of the specific ligand binding sites are also relevant to "RNA world" models of the origin of life, in which the evolution of intermediary metabolism occured at the time of the RNA world. Finally, determination of these structures should be useful in the design of new catalysts which need these co-factors for their function. ***

9506913 Feigon Aptamer是为DNA或RNA寡核苷酸创造的名称，这些名称是从含有随机核苷酸序列区域的大量寡核苷酸中选择的，用于与特定靶分子结合。 隔离过程涉及对特定靶标有亲和力的寡核苷酸的重复选择和富集的循环，然后使用聚合酶链反应（PCR）扩增这些序列。 最终通过克隆和测序来鉴定具有所选特征，即与特定分子结合的橄榄核苷酸，即与特定分子结合。 通常对克隆序列进行筛选以获得共同的共识和可能的次要结构基序。 该方法已用于识别许多不同类别的RNA适体，它们专门与常见的代谢底物和辅助因子结合。 这些包括ATP，GTP，核黄素，FAD，FMN，氰callamin和氨基酸精氨酸和瓜氨酸。 基于共识序列和进一步的实验，例如缺失分析，在大多数情况下，已经确定了最小的结合序列，并提出了二次结构。 但是，迄今为止，尚未确定RNA适体的三维结构。 多维多核NMR光谱将用于确定在没有结合辅助因子的情况下结合生物辅因子的适体的三维结构。 使用T7 RNA聚合酶在体外转录中将合成用于NMR研究的RNA样品。 这些最初将通过1H NMR光谱进行研究，以筛选提供良好NMR光谱的序列。 一旦获得了良好的样本，将合成部分或均匀的15N和/或13C标记的样品，以促进完全共振分配。 在有利的情况下，将使用度量矩阵距离几何形状和通过分子动力学进行完整确定完整的三维结构。 NMR光谱法将用于研究结合生物辅助因子的RNA寡核苷酸的结构。 这些RNA包含各种建议的二级结构基序，包括发夹环，内环，伪诺和分子内G-四分之一。 因此，确定其结构将使人们对核酸折叠的原理有更深入的了解。 到目前为止，很少有任何类型的RNA结构已解决，因此有关RNA三级结构的任何信息都应大大增加这些二级结构基序的知识库。 确定特定辅因子RNA相互作用还将有助于阐明蛋白质和其他配体对RNA的特定识别的某些因素。 特定配体结合位点的结构也与生命起源的“ RNA世界”模型有关，其中中介代谢在RNA世界时发生了。 最后，确定这些结构应该在设计新催化剂的设计中很有用，这些催化剂需要这些co因子的功能。 ***