FUNCTIONALLY TETHERED OLIGONUCLEOTIDES

在超卷或超卷引起的扭应力的影响下 蛋白质的结合，DNA可以被强迫采用各种结构 基本上偏离了Watson和Watson的规范B型双链 克里克。尽管DNA的这种结构可塑性深深地交织在一起 由于它的生物学功能，非规范DNA的许多方面仍然存在 人们对此知之甚少。这项计划的一个主要目标，广义地说，是 阐明DNA结构如何适应扭转应力，以确定 各种失真的能量成本，并探讨 工程扭曲对蛋白质识别DNA的影响。其中之一 缺乏关于非规范的信息的主要原因 结构通常只在存在的情况下形成 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。建议数 研究ALM通过使用二硫键交联来克服这一问题 在合成中设计特定类型的非正则结构 寡核苷酸。这些经过改造的分子将被用来研究 关于结构、能量学和蛋白质的识别。 前面的研究提供了一个例子，说明了合成改性是如何 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。与众多方法形成对比的是 当前可用于特定于站点的系留功能连接 对于DNA来说，相对较少的方法可以引入相同的 对RNA的各种修饰。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 过程中，对操纵RNA结构的方法的需求变得前所未有 更敏锐。拟议研究的另一个方面将解决这一需要。 也就是说，将发展化学以允许特定部位的附着 通过可转换的核苷将功能拴在RNA上 接近。这些功能拴系的几个应用 将探索的寡核苷酸：(I)高容量的合成 含有HIV-L焦油元件的核糖核酸亲和柱，将用于 人类免疫缺陷病毒宿主RNA结合因子的纯化 转录激活；和(Ii)寡核苷酸的合成 包含放置在5‘-剪接点内的系链光活性基团 为了通过光亲和标记法进行鉴定 参与RNA剪接的蛋白质因子。

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.