FUNCTIONALLY TETHERED OLIGONUCLEOTIDES

功能性连接的寡核苷酸

• 批准号: 2182812
• 负责人: GREGORY Lawrence VERDINE
• 金额: $ 27.9万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-31 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182812
• 关键词: DNA; DNA binding protein; RNA binding protein; affinity chromatography; affinity labeling; chemical structure function; disulfide bond; human immunodeficiency virus 1; intermolecular interaction; messenger RNA; nucleic acid chemical synthesis; nucleic acid probes; nucleic acid sequence; nucleic acid structure; nucleotide analog; oligonucleotides; photoactivation; polarimetry; synthetic nucleic acid; virus RNA

Under the influence of torsional stress induced by supercoiling or the binding of proteins, DNA can be coerced to adopt a variety of structures that deviate substantially from the canonical B-form duplex of Watson and Crick. Although this structural plasticity of DNA is deeply intertwined with its biological function, many facets of non-canonical DNA remain poorly understood. A primary goal of this program, broadly stated, is to elucidate how DNA structure accommodates torsional stress, to determine the energetic costs of various kinds of distortion, and to probe the effects of engineered distortions on DNA recognition by proteins. One of the principal reasons for the lack of information on non-canonical structures has been that they are generally formed only in the presence of complex macromolecular assemblies, and hence they have been difficult to embody in small, structurally characterizeable systems. The proposed studies alm to overcome this problem by use of disulfide cross-linking to engineer specific kinds of non-canonical structures in synthetic oligonucleotides. These engineered molecules will then be studied with regard to structure, energetics, and recognition by proteins. The foregoing studies provide an example of how synthetic modification of DNA can be used to gain information on its structure, function, and interaction with other macromolecules. In contrast to the numerous methods currently available for site-specific attachment of tethered functionality to DNA, relatively few methods are available for introducing the same sorts of modification into RNA. As it becomes increasingly apparent that RNA-protein interactions control a wide range of important biological processes, the need for methods to manipulate RNA structure becomes ever more acute. Another aspect of the proposed studies will address this need. Namely, chemistry will be developed to permit the site-specific attachment of tethered functionality to RNA through the convertible nucleoside approach. Several applications of these functionally tethered oligoribonucleotides will be explored: (i) synthesis of a high-capacity RNA affinity column containing the TAR element of HIV- l, to be used in purification of host RNA-binding factors that contribute to HIV transcriptional activation; and (ii) synthesis of oligoribonucleotides containing a tethered photoactive group placed within the 5'-splice site of a eukaryotic mRNA, in order to identify through photoaffinity labelling protein factors that participate in RNA splicing.

在过卷或超卷引起的扭转应力的影响下， 结合蛋白质，DNA可以被迫采取各种结构 其实质上偏离沃森的规范B型双链体， 克里克尽管DNA的这种结构可塑性 由于其生物学功能，非规范DNA的许多方面仍然存在， 不太了解。从广义上讲，该计划的主要目标是 阐明DNA结构如何适应扭转应力，以确定 各种扭曲的能量成本，并探讨 工程扭曲对蛋白质识别DNA的影响。之一 缺乏非典型信息的主要原因 结构的一个重要特征是，它们通常仅在存在下形成， 复杂的大分子组装，因此它们很难 体现在小的、结构上可表征的系统中。拟议 研究了通过使用二硫化物交联来克服这个问题， 在合成中设计特定种类的非规范结构 寡核苷酸这些工程分子将被研究， 关于结构、能量学和蛋白质识别。 上述研究提供了如何合成修饰的实例。 DNA可以用来获得有关其结构、功能和 与其他大分子的相互作用。与众多的方法相比， 目前可用于特定位置的系留功能连接 对于DNA，相对较少的方法可用于引入相同的 对RNA进行修饰。随着越来越明显的是， RNA-蛋白质相互作用控制着广泛的重要生物学过程， 随着这些过程的发展，对操纵RNA结构的方法的需求变得越来越大。 越来越紧张拟议研究的另一个方面将解决这一需要。 也就是说，化学将被开发以允许位点特异性附着 通过可转化的核苷将功能性连接到RNA上， approach.这些功能性连接的几个应用程序 将探索寡核糖核苷酸：（i）合成高容量的 含有HIV-1的TAR元件的RNA亲和柱，用于 有助于HIV的宿主RNA结合因子的纯化 转录激活;和（ii）寡核糖核苷酸的合成 含有位于5 ′-剪接位点内的系留光敏基团 为了通过光亲和标记进行鉴定， 参与RNA剪接的蛋白质因子。