Site-Specific Recognition Restriction Enzymes

描述（由申请人提供）：我们将开发一种用于体外和体内应用的双链DNA位点特异性切割的新方法。这种方法将创造出一类新的DNA限制酶，研究人员可以设计这种酶来特异性地识别基因组或其他大片段DNA中的20个碱基对或更长的独特序列。这些新的酶将成为体外和体内切割全基因组DNA的有力工具。它们将允许精确控制诸如“敲除”基因实验的过程，促进真核细胞中的同源重组，并且能够在体外出于各种目的（包括功能基因组学工作）对大DNA进行再工程化和/或克隆。新方法涉及两组元素。第一种是生物素化的靶向寡核苷酸（TON），其与待切割的DNA的两条链上的序列同源。TON将在recA蛋白存在下与靶DNA一起孵育，这有助于形成“D -环”复合物.在这些D环中，TON与双链DNA内的精确识别序列杂交，仅在该区域中置换DNA的互补链。该方案中的第二个元件是Fok I的催化亚基，Fok I是一种IIS型限制性内切酶，已显示其在结合时切割DNA（3，4，5）。我们将对Fok I的N末端（Fn）催化亚基进行基因工程改造，使其与E. coli生物素化肽。这个序列导致E.大肠杆菌中，以使与其融合的表达蛋白生物解酶化。这种融合的Fn蛋白仅在与链霉亲和素结合成复合物时才切割DNA，链霉亲和素又与上述杂交的生物素化TON结合。该方案的识别和切割元件的组合将产生允许在精确选择的位点切割DNA的“设计者”限制酶。在第一阶段之前，我们成功地克隆和表达了工程化的FokI（Fn）蛋白，并显示其具有酶活性。用质粒模板证明recA介导的TON插入反应。在第一阶段，我们将提高生物素化Fn酶的表达和纯化，并测量recA介导的TON与大片段DNA的结合。将测试这两种组分以确定它们是否特异性切割大DNA。由此产生的产品，如试剂盒和服务，适用于全球45，000个分子生物学实验室。

DESCRIPTION (provided by applicant): We will develop a novel method for site-specific cleavage of double-stranded DNA for in vitro and in vivo applications. This method will create a new class of DNA restriction enzymes that can be designed by the researcher to specifically recognize a 20 base pair or longer unique sequences within a genome or other large stretch of DNA. These new enzymes will become powerful tools for in vitro and in vivo cleavage of whole genome DNAs. They will allow precise control over processes such as "knock-out" gene experiments, facilitating homologous recombination in eukaryotic cells, and enabling re-engineering and/or cloning of large DNAs in vitro for various purposes, including functional genomics work. The novel method involves two sets of elements. The first are biotinylated targeting oligonucleotides (TONs) that are homologous to the sequences on both strands of the DNA to be cleaved. The TONs will be incubated with the target DNA in the presence of recA protein, which facilitates the formation of "D -loop" complexes. In these D-loops the TONs are hybridized to exact recognition sequences within the double-stranded DNA, displacing the complementary strand of the DNA only in that region. The second element in this scheme is the catalytic subunit of Fok I, a type IIS restriction enzyme, which has been shown to cleave DNA when bound (3,4,5). We will genetically engineer the Fok I N-terminal (Fn) catalytic subunit so that it will be fused to an E. coli biotinylation peptide at one of its ends. This sequence causes E. coli to bioltinylate the expressed protein to which it is fused. This fused Fn protein will cleave DNA only when bound in a complex with streptavidin that is in turn bound to the hybridized biotinylated TONs described above. The combination of the recognition and cleavage elements of this scheme will create "designer" restriction enzymes that will allow cleavage of DNA at precisely selected sites. Prior to Phase I, we successfully cloned and expressed the engineered Fokl (Fn) protein and showed that it is enzymatically active. The recA mediated TON insertion reaction was demonstrated with a plasmid template. In Phase I, we will improved the expression and purification of the biotinylated Fn enzyme, and measure the recA-mediated binding of the TONs to large segments of DNA. The two components will be tested to determine if they specifically cleave large DNAs. The resulting products, as kits and services, are applicable to 45,000 molecular biology labs worldwide.