COMPLEXES OF DNA WITH PEPTIDE NUCLEIC ACID (PNA)

这一建议的目的是研究一种新的 具有潜在吸引力的药物，肽核酸(PNA)，具有双- 搁浅(DS)DNA。将对药物的独特性质进行调查， 它包含了异常强大和高度特异的序列 通过链置换反应与dsDNA结合。对此进行全面的研究 靶向dsDNA的新模式，将开发特殊的分析方法。这个 电泳迁移率变化分析将基于不同的迁移率 在双链DNA片段的聚丙烯酰胺凝胶电泳法过程中 及其与PNA的复合体。核酸酶切割试验将基于 单链特异性核酸酶切割dsDNA的能力 PNA与dsDNA结合的位置。电子显微镜化验将使用 生物素标记的PNA和链霉亲和素作为PNA部位的标记 与dsDNA相关。扫描力显微镜将用于研究 PNA/dsDNA复合体的精细结构。 这些方法将用于定量研究水合物的动力学。 不同长度和长度的PNA低聚体的链置换反应 作为环境条件的函数的序列。BIS-PNA，由两个 由柔性连接物连接的PNA低聚物，带有赖氨酸的PNA 不同长度，插入剂标记的PNA，DNA-PNA偶联物，DNA-双- PNA偶联物，PNA包含特殊设计的碱基类似物和许多 其他PNA衍生品也将被研究。累积的数据将是 用来建立链的详细理论模型-- 置换反应。模型中的关键点是PNA~NA 嘌呤DNA链和两个高嘧啶PNA分子之间的三链(或 BIS-PNA的两半)。DNA~PNA的解离(熔融)动力学 将研究各种模型体系的络合物，如DNA/PNA 双链和三链，以确定基本的动力学参数。 广泛的理论模型的过程中的各种途径 链的位移将由蒙特卡罗方法进行。 理论建模的许多结果将被测试，并且 模型将进行调整，以满足整个实验数据。这个 该模型不仅适用于高嘧啶PNA/dsDNA复合体，而且适用于高嘧啶PNA/dsDNA复合体。 此外，还将对同型嘌呤PNA/dsDNA复合体进行测试。这个 PNA/dsDNA相互作用的序列特异性与 反应的机理将会被确定。 从基本观点来看，该项目的主要成果将是 了解一种全新的DNA靶向模式。关键字 将回答关于决定强势和强势的因素 PNA与DNA的序列特异性结合。在实践中，少校 该项目的成果将在于找到提高效率的方法 以及PNA类药物的序列特异性。所阐述的方法将 使筛选各种PNA类似物成为可能 在基因组中定位特定位置的能力。尤其是多聚嘌呤 HTV-I基因组中的区域将以双链DNA的形式和在 DNA/RNA异源双链的形式。

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.