HYBRID ENDONUCLEASES FOR GENOMIC RESEARCH AND ANALYSIS

用于基因组研究和分析的混合核酸内切酶

• 批准号: 6386252
• 负责人: SRINIVASAN CHANDRASEGARAN
• 金额: $ 27.68万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386252
• 关键词: DNA binding protein; biomedical resource; chimeric proteins; computer simulation; conformation; gene therapy; genetic library; genome; high performance liquid chromatography; hybrid enzyme; laboratory mouse; microinjections; molecular cloning; nuclear magnetic resonance spectroscopy; oligonucleotides; polymerase chain reaction; protein engineering; protein protein interaction; protein structure; restriction endonucleases

DESCRIPTION (Adapted from the Investigator's Abstract): Chimeric restriction enzymes are a novel class of engineered nucleases in which the nonspecific DNA cleavage domain (a type IIs restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely, the zinc finger motif, the helix-turn-helix motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by micro-injection of DNA substrates and the enzyme into frog oocytes. The injected enzyme made site-specific double-strand breaks in the targets even after assembly of DNA into chromatin. In addition, this cleavage activated the target modules for efficient homologous recombination. The goal here is to redesign and improve the chimeric nucleases by computer modeling based on the crystal structures of native FokI and FokI bound to DNA. The investigators also plan to increase the sequence specificity of the hybrid endonucleases by using polydactyl zinc fingers. They plan to induce homologous recombination at a chromosomal site by using engineered chimeric nucleases within a human or animal cell line. Their long term goal is the application of chimeric nucleases to correct a genetic defect in an animal model.

描述（改编自研究者的摘要）：嵌合限制 酶是一类新型的工程核酸酶，其中非特异性 DNA 切割结构域（II 型限制性内切核酸酶）与其他结构域融合 DNA 结合基序。后者包括三种常见的真核DNA结合 基序，即锌指基序、螺旋-转角-螺旋基序和基本基序 含有亮氨酸拉链基序的螺旋-环-螺旋蛋白。这样的嵌合 核酸酶已被证明可以在体外进行非常接近的特异性切割 预期的识别序列。最重要的嵌合核酸酶是那些 基于锌指 DNA 结合蛋白，因为其模块化结构。 最近，一种这样的嵌合核酸酶 Zif-QQR-F(N) 被证明可以发现并切割 它的体内目标。这是通过 DNA 底物的显微注射进行测试的 将酶转化为青蛙卵母细胞。注射的酶具有位点特异性 即使在 DNA 组装成染色质后，目标中也会出现双链断裂。 此外，这种裂解激活了目标模块，以实现高效 同源重组。这里的目标是重新设计和改进嵌合体 基于天然 FokI 晶体结构的计算机建模核酸酶 FokI 与 DNA 结合。研究人员还计划增加序列 通过使用多指锌指来确定杂合核酸内切酶的特异性。他们 计划通过使用在染色体位点诱导同源重组 人类或动物细胞系内的工程嵌合核酸酶。他们的长 术语目标是应用嵌合核酸酶来纠正遗传缺陷 在动物模型中。