Recognition of DNA by Modified Peptide Nucleic Acids (PNA) for Gene Targeting and Analysis

我们提出了一项旨在通过PNA进行基因分析和基因特异性靶向的新化学和新技术的研究。DNA模拟多肽核酸在遗传诊断、分子生物学和药物开发中得到了广泛的应用。这是由于PNA具有非常好的DNA和RNA识别特性以及非常高的生物(和化学)稳定性，以及“化学灵活性”。因此，PNA齐聚物可以根据不同的应用“量身定做”。在这个项目中，我们打算探索和开发用于基因检测和靶向的PNA化学修饰和偶联物。具体地说，我们计划设计和合成新的碱基，用于通过三链形成和螺旋入侵对双链DNA进行序列特异性识别。这些PNA寡聚体将以Ha-ras和c-myc癌基因为模型基因，在细胞基因靶向(反义和反基因)分析中进行分析。此外，还将制备PNA与脂类、多肽、荧光团和DNA嵌入剂的偶联物，并对其DNA识别、细胞摄取和(反义和反基因)基因敲除活性进行表征。用X射线结晶学方法研究了新型PNA齐聚物与DNA和PNA形成的配合物的结构。最后，将利用定制制造PNA低聚物的能力来设计和分析PNA捕获和检测探针，以开发以杂交阵列形式在线、固体支持的DNA分子分析。

We propose a study aimed at novel chemistry and technology for gene analysis and gene specific targeting by PNA. The DNA mimic PNA (peptide nucleic acid) is finding widespread applications within genetic diagnostics, molecular biology and drug discovery. This is due to the very favourable DNA and RNA recognition properties of PNA combined with very high biological (and chemical) stability, as well as "chemical flexibility". Thus PNA oligomers can be "tailor made" to a variety of applications. In this project we propose to explore and exploit PNA chemical modifications and conjugates for gene detection and targeting. Specifically, we plan to design and synthesize novel nucleobases for sequence specific recognition of duplex DNA by triplex formation and helix invasion. Such PNA oligomers will be analysed in cellular gene targeting (antisense and antigene) assays using the Ha-ras and c-myc oncogenes as model genes. Furthermore, conjugates of PNA with lipids, peptides, fluorophores and DNA intercalators will be prepared and characterized in terms of DNA recognition, cellular uptake and (antisense and antigene) gene knock down activity. The structure of novel PNA oligomer complexes with DNA and PNA will be studied by X-ray crystallography. Finally, the capacity to tailor make PNA oligomers will be exploited to design and analyse PNA capture and detection probes for development of novel on line, solid support analysis of DNA molecules in hybridisation array formats.