SITE SPECIFIC CHEMICAL METHODS FOR CLEAVING GENOMIC DNA

总体目标是提供化学方法， 基因组DNA的序列特异性切割， 一系列新的特异性，其序列特异性高于 是目前可用的限制酶。 嘧啶寡核苷酸特异性结合双链DNA序列 形成三螺旋结构。 嘧啶 寡聚体在DNA的大沟中平行于 沃森-克里克嘌呤链Hoogsteen碱基配对。 寡核苷酸，长度为15-18个碱基，并配备有DNA 在5'端切割功能EDTA-Fe，导致序列特异性 质粒DNA中单个位点的双链断裂4 kbp， 噬菌体基因组全长（48.5kbp）。 由于长度的 在正式意义上，这是106倍以上 比限制性内切酶更特异的序列。 重要的是要 确定这种切割特异性的全部潜力 可以实现 因为修饰的寡核苷酸可以是 通过自动化方法合成，这种方法提供了一种新的 一类“稀有切割机”，这可能成为容易获得的 生物群落 具体目标是（1）优化 通过寡核苷酸的合成和研究切割效率 连接有多个EDTA-Fe部分的探针，（2）分析 通过研究寡核苷酸结合的序列特异性， pH、温度和有机共溶剂对探针影响 不同的基组成，（3）推广了一组条件， 在长度为15-18个碱基对的嘌呤-DNA位点上的独特识别， (4)定义用于定位部分同源性的DNA位点的条件， (5)分析DNA靶大小对DNA分子动力学的影响， 结合并测量离散位点的结合亲和力，（6） 制定提高特异性与非特异性比率的方法 通过研究基于寡核苷酸的协同结合 碱基堆积和肽-DNA探针，（7）设计和合成 用于混合嘌呤-嘧啶序列的新的寡核苷酸，（8） 构建新的“交叉”寡核苷酸用于结合 回文同嘌呤序列，（9）发生后裂解 在基因组DNA的特异性EDTA切割位点的标记方法， (10)研究切割后标记是否可用于DNA 通过凹端杂交特异性片段。

The overall objective is to provide chemical methods for the sequence specific cleavage of genomic DNA which afford a broad range of new specificities with higher sequence specificity than is currently available with restriction enzymes. Pyrimidine oligonucleotides bind duplex DNA sequence specifically at purine sites to form a triple helix structure. The pyrimidine oligomer is oriented in the major groove of DNA parallel to the Watson-Crick purine strand by Hoogsteen base pairing. Oligonucleotides, 15-18 bases in length, and equipped with a DNA cleaving function EDTA-Fe at the 5' end, cause sequence specific double strand breaks at single sites in plasmid DNA 4 kbp and lambdal bacteriophage genome (48.5 kbp). Due to the length of the recognition site, in a formal sense, this is 106 times more sequence specific than restriction enzymes. It is important to determine whether the full potential of this cleavage specificity can be realized. Because modified oligonucleotides can be synthesized by automated methods, this methodology affords a new class of "rare cutters" which could become readily available to the biological community. Specific objectives are to (1) optimize cleavage efficiency by the synthesis and study of oligonucleotide probes with multiple EDTA-Fe moieties attached, (2) analyze sequence specificity of oligonucleotide binding by studying the influence of pH, temperature, and organic cosolvent on probes of different base composition, (3) generalize a set of conditions for unique recognition at purine-DNA sites 15-18 base pairs in length, (4) define conditions for mapping DNA sites of partial homology, (5) analyze the effects of DNA target size on the kinetics of binding and measure binding affinities at discrete sites, (6) develop methods for raising the ratio of specific to non-specific binding by studying cooperatively binding oligonucleotides based on base stacking, and peptide-DNA probes, (7) design and synthesize novel oligonucleotides for mixed purine-pyrimidine sequences, (8) construct novel "crossover" oligonucleotides for binding palindromic homopurine sequences, (9) develop post-cleavage labeling methods at specific EDTA cleavage sites of genomic DNA, (10) investigate whether post-cleavage labeling could be made DNA fragment specific by recessed end hybridization.