NEW CLASS OF GENOME RARE CUTTERS

描述（改编自研究者摘要）： 该项目包括开发一种切割基因组DNA的新策略， 将最常见的限制性内切酶转化为稀有和超稀有的 切割器产生0.1-10 Mbp范围内的DNA片段。主要工具为 达到这一目标的是肽核酸（PNA），一种合成的模拟肽核酸。 寡核苷酸，其携带连接到聚酰胺骨架的DNA碱基。 高嘧啶PNA形成非常强和高度序列特异性的复合物 双链DNA 在这个称为P环的复合物中，两个同型嘧啶PNA 分子与互补的嘌呤链形成三链体， 嘧啶DNA链被置换。 该项目的一个主要想法是 序列特异性PNA与相对较短的DNA序列结合， DNA识别甲基转移酶（甲基化酶），如果PNA结合位点 与甲基化酶结合位点重叠。 如果这是真的， 足够的序列特异性，这将开启 应用PNA切割基因组DNA：甲基化后， 在PNA/DNA复合物中，去除PNA，并通过适当的酶切DNA。 限制性内切酶仅在被保护免于甲基化的位点 的PNA。 为了达到该项目的目标，PNA/DNA复合物的形成， 将详细研究各种PNA修饰。 的动力学和 由两个PNA组成的bis-PNA与双链DNA的相互作用机制 通过柔性接头/双PNA连接的分子， 正电荷和其他PNA修饰，将进行研究。 基于 详细了解P环形成的机制， 选择合适的PNA修饰用于基因组切割。 的 甲基化酶与PNA/DNA复合物相互作用的机理将被研究 选择PNA阻止位点甲基化的条件， 由于PNA结合而受到保护。 一个假设将被测试，甲基化酶 显著地增强了在完美和 不完美的PNA结合，因为不完美的PNA/DNA复合物不能阻止 甲基化 不同的PNA结构将在几个基因组DNA上进行测试， 与不同的甲基化-限制酶对组合。 的 该项目的主要成果将包括开发一个新的巨大的 一类基因组切割器，选择性地切割各种基因组DNA， 长度范围，从每100 kbp一个到每1 Mbp一个，甚至更少，如果 必要 这是目前很少有切割机的范围，如果有的话， available. 这可以显著地简化本发明中的许多技术。 整个DNA分析领域，包括基因组作图、克隆和 测序

DESCRIPTION (Adapted from the Investigator's Abstract): The goal of the project consists in developing a new strategy of cutting genomic DNA, which converts the most common restriction enzymes into rare and super-rare cutters yielding DNA fragments in the range 0.1-10 Mbp. The major tool for reaching the goal is Peptide Nucleic Acid (PNA), a synthetic mimic of oligonucleotides, which carries DNA bases attached to a polyamide backbone. Homopyrimidine PNAs form a very strong and highly sequence-specific complex with duplex DNA. In this complex, called P-loop, two homopyrimidine PNA molecules form a triplex with the complementary purine strand leaving the pyrimidine DNA strand displaced. A major idea underlying the project is that sequence-specific PNA binding to relatively short DNA sequences blocks DNA recognition by methyltransferase (methylase) if the PNA binding site overlaps with the methylase binding site. If this were true and PNA binding were sufficiently sequence-specific, it would open the possibility of applying PNA for the cleavage of genomic DNA: after methylation of a PNA/DNA complex, PNA is removed and DNA is cleaved by an appropriate restriction enzyme only at the sites that were protected against methylation by PNA. To reach the objectives of the project, the PNA/DNA complex formation for various PNA modifications will be studied in great detail. The kinetics and mechanism of interaction with duplex DNA of bis-PNA, consisting of two PNA molecules connected by a flexible linker/bis-PNA carrying additional positive charges and other PNA modifications, will be studied. Based on the detailed understanding of the mechanism of the P-loop formation, the most appropriate PNA modification will be chosen for genome cutting. The mechanism of interaction of methylases with PNA/DNA complex will be studied to select conditions under which PNA prevents methylation of the sites protected due to PNA binding. A hypothesis will be tested that methylase significantly enhances the selectivity of discrimination between perfect and imperfect PNA binding because imperfect PNA/DNA complexes do not prevent methylation. Different PNA constructions will be tested on several genomic DNAs in combination with different methylation-restriction enzymatic pairs. The major result of the project will consist in the development of a new huge class of genome cutters, which selectively cut genomic DNAs in various ranges of lengths, from one per 100 kbp to one per 1 Mbp and even rarer, if necessary. This is the range where very few cutters, if any, are presently available. This could significantly simplify numerous techniques in the entire field of DNA analysis, including genome mapping, cloning and sequencing.