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费贡适体是从含有与特定靶分子结合的随机核苷酸序列区域的大量寡核苷酸中挑选出来的脱氧核糖核酸或核糖核酸的名称。分离过程包括重复选择和浓缩与特定靶标有亲和力的寡核苷酸，然后使用聚合酶链式反应(PCR)扩增这些序列。通过克隆和测序，最终鉴定出具有选定特征的寡核苷酸，即与特定分子结合。通常对克隆的序列进行筛选，以寻找共同的共识和可能的二级结构基序。这一方法已经被用来鉴定一些不同类别的RNA适配子，它们专门与共同的代谢底物和辅因子结合。这些物质包括ATP、GTP、核黄素、FAD、FMN、氰基钴胺以及氨基酸精氨酸和瓜氨酸。基于一致序列和进一步的实验，如缺失分析，在大多数情况下已经确定了最小结合序列，并提出了二级结构。然而，到目前为止，还没有确定RNA适配子的三维结构。多维多核核磁共振波谱将被用来确定结合生物辅因子的适配子的三维结构，在没有和存在结合辅因子的情况下。用于核磁共振研究的RNA样品将通过使用T7 RNA聚合酶体外转录来合成。这些将首先通过1H核磁共振波谱进行研究，以筛选出具有良好核磁共振谱的序列。一旦获得性能良好的样品，将部分或均匀地合成15N和/或13C标记的样品，以便于完成共振分配。在有利的情况下，完整的三维结构将使用度量矩阵距离几何和分子动力学的精化来确定。核磁共振波谱将用于研究与生物辅因子结合的RNA寡核苷酸的结构。这些RNA包含各种各样的提议的二级结构基序，包括发夹环、内部环、假结和分子内G-四元组。因此，确定它们的结构将有助于更好地理解核酸折叠的原理。到目前为止，很少有任何类型的RNA结构被解决，所以任何关于RNA三级结构的信息都应该大大增加这些二级结构基序的知识库。确定特定的辅因子-RNA相互作用也将有助于阐明蛋白质和其他配体对RNA的序列特异性识别的一些因素。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，在该模型中，中间代谢的进化发生在RNA世界的时间段。最后，对这些结构的测定应该有助于设计需要这些辅助因子来发挥其功能的新催化剂。***