NEW CLASS OF GENOME RARE CUTTERS

描述(改编自《调查员摘要》)： 该项目包括开发一种新的切割基因组DNA的策略， 将最常见的限制性内切酶转化为稀有和超稀有 切割器产生0.1-10 MBP范围的DNA片段。主要的工具是 达到这一目标的是肽核酸(PNA)，它是一种合成的 寡核苷酸，它携带连接到聚酰胺主干上的DNA碱基。 高嘧啶核糖核酸形成一种非常强的高度序列特异性的复合体 带有双链DNA。在这个被称为P-环的复合体中，两个高嘧啶PNA 分子与互补的嘌呤链形成三链，留下 嘧啶脱氧核糖核酸链置换。该项目背后的一个主要想法是 序列特异的PNA与相对较短的DNA序列结合阻止 甲基转移酶(甲基酶)识别DNA是否与PNA结合 与甲基酶结合部位重叠。如果这是真的，且PNA具有约束力 如果有足够的序列特异性，它将开启一种可能性 应用PNA进行基因组DNA切割：A基因甲基化后 PNA/DNA复合体，PNA被去除，DNA被适当的 限制性内切酶仅在防止甲基化的位置 被巴勒斯坦民族权力机构。 为了实现该项目的目标，PNA/DNA复合体的形成 将非常详细地研究对PNA的各种修改。动力学和 双链PNA与双链DNA相互作用机制的研究 通过柔性接头/双PNA连接的分子携带额外的 正电荷和其他PNA修饰，将被研究。基于 详细了解P环的形成机理，最 将选择适当的PNA修饰进行基因组切割。这个 将研究甲基酶与PNA/DNA复合体的相互作用机制 选择PNA阻止位点甲基化的条件 由于PNA绑定而受到保护。将检验一种假设，即甲基酶 显著提高了区分完美和非完美之间的选择性 不完美的PNA结合，因为不完美的PNA/DNA复合体不能阻止 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-限制性内切酶组合。这个 该项目的主要成果将在于开发一个新的巨型 一类基因组切割器，它选择性地切割各种不同的基因组DNA 长度范围，从每100 KBP一个到每1 MBP一个，甚至更罕见，如果 这是必要的。这是目前极少有切割机的范围。 可用。这可以大大简化 整个DNA分析领域，包括基因组测绘、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.