COMPLEXES OF DNA WITH PEPTIDE NUCLEIC ACID (PNA)

本提案的目的是研究一种新的 肽核酸（PNA）是一种具有潜在吸引力的药物， 双链DNA（ds DNA）将研究药物的独特性质， 其包括异常强和高度序列特异性 通过链置换反应与dsDNA结合。完全研究这个 针对dsDNA的新模式，将开发特殊的检测方法。的 电泳迁移率变动分析将基于不同的迁移率 在dsDNA片段的聚丙烯酰胺凝胶电泳过程中， 及其与PNA的复合物。核酸酶切割测定将基于 单链特异性核酸酶在位点切割dsDNA的能力 其中PNA与dsDNA结合。电子显微镜分析将使用 生物素化的PNA和作为PNA位点标记的链霉亲和素 与dsDNA的关系扫描力显微镜将用于研究 PNA/dsDNA复合物的精细结构。 这些方法将用于定量研究的动力学的 不同长度PNA寡聚体的链置换反应， 序列作为环境条件的函数。Bis-PNA，由两个 通过柔性接头连接的PNA寡聚体，具有赖氨酸“尾”的PNA， 不同长度，嵌入剂标记的PNA，DNA-PNA缀合物，DNA-双- PNA缀合物，含有特别设计的碱基类似物的PNA和许多 还将研究其它PNA衍生物。积累的数据将是 用于制定详细的理论模型的股- 位移反应模型中的关键点将是PNA~NA 嘌呤DNA链和两个同型嘧啶PNA分子之间的三链体（或 两半双PNA）。DNA~PNA解离（熔解）动力学 复合物将研究各种模型系统，如DNA/PNA 双链体和三链体，以确定基本的动力学参数。 广泛的理论建模的过程中的各种途径， 通过蒙特卡罗方法进行链置换。 理论模型的许多结果将被测试， 模型将进行调整，以满足整个身体的实验数据。的 该模型不仅适用于同型嘧啶PNA/dsDNA复合物， 还将测试高嘌呤PNA/dsDNA复合物。的 PNA/dsDNA相互作用的序列特异性与 反应的机理将被建立。 从基本观点来看，该项目的主要成果将是 这是一种全新的DNA靶向模式。枢转 问题将得到回答的因素，决定双方的强大和 PNA与DNA的序列特异性结合。实际上，主要 该项目的成果将包括找到提高效率的方法 和序列特异性。所阐述的方法将 使其有可能筛选各种PNA类似物， 靶向基因组特定位点的能力。特别是，聚嘌呤 HTV-I基因组中的片段将以双链DNA的形式被靶向， DNA/RNA异源双链体的形式。

The goal of this proposal is to study the mechanism o interaction o a new potentially attractive drug, Peptide Nucleic Acid (PNA), with double- stranded (ds) DNA. The unique property of the drug will be investigated, which consists in exceptionally strong and highly sequence-specific binding to dsDNA via strand displacement reaction. To study this totally new mode of targeting dsDNA, special assays will be developed. The electrophoretic mobility shift assay will be based on different mobility in the course of polyacrylamide gel electrophoresis of a dsDNA fragment and its complex with PNA. The nuclease cutting assay will be based on the ability of single strand-specific nucleases to cut dsDNA at the sites where PNA binds to dsDNA. The electron-microscopy assay will use biotinylated PNA and streptavidin as a marker of the sites of the PNA association with dsDNA. Scanning force microscopy will be used to study the fine structure of PNA/dsDNA complexes. These methods will be used for quantitative studies of the kinetics of the strand displacement reaction for PNA oligomers of different length and sequence as a function of ambient conditions. Bis-PNA, consisting of two PNA oligomers connected by a flexible linker, PNAs with lysine "tails" of different length, intercalator-tagged PNAs, DNA-PNA conjugates, DNA-bis- PNA conjugates, PNA containing specially designed base analogs and many other PNA derivatives will also be studied. The accumulated data will be used to formulate the detailed theoretical model of the strand- displacement reaction. The key point in the model will be the PNA~NA triplex between purine DNA strand and two homopyrimidine PNA molecules (or two halves of bis-PNA). Dissociation (melting) kinetics of DNA~PNA complexes will be studied for various model systems, such as DNA/PNA duplexes and triplexes, to determine fundamental kinetic parameters. Extensive theoretical modeling of the various pathways of the process of the strand displacement will be performed by the Monte Carlo method. Numerous consequences of the theoretical modeling will be tested and the model will be adjusted to satisfy the whole body of experimental data. The validity of the model not only for homopyrimidine PNA/dsDNA complexes but also for homopurine PNA/dsDNA complexes will be also tested. The correlation between the sequence specificity of PNA/dsDNA interaction and the mechanism of the reaction will be established. The major result of the project from the basic viewpoint will be understanding of an entirely new mode of DNA targeting. The pivotal question will be answered about the factors determining both strong and sequence specific binding of PNA with DNA. In practical terms, the major outcome of the project will consist in finding ways to improve efficiency and sequence specificity of PNA-like drugs. The methods elaborated will make it possible to screen various PNA analogs with respect to their ability to target specific sites at genomes. In particular, the polypurine tract in HTV-I genome will be targeted in the form of duplex DNA and in the form of a DNA/RNA heteroduplex.