Site-Specific Recognition Restriction Enzymes

描述(申请人提供)：我们将开发一种新的方法来定点切割双链DNA，用于体外和体内应用。这种方法将创造一种新的DNA限制性内切酶，研究人员可以设计这种酶来专门识别基因组或其他大片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的强大工具。它们将允许对“敲除”基因实验等过程进行精确控制，促进真核细胞中的同源重组，并能够在体外重新设计和/或克隆用于各种目的的大DNA，包括功能基因组学工作。新的方法涉及两组元素。第一种是生物素化的靶向寡核苷酸(TON)，这些寡核苷酸与要切割的DNA的两条链上的序列同源。在recA蛋白存在的情况下，Tons将与目标DNA孵育，这有助于形成“D-loop”复合体。在这些D-环中，启动子与双链DNA中的准确识别序列杂交，仅在该区域取代DNA的互补链。该方案中的第二个元件是FOK I的催化亚单位，它是一种IIS限制性内切酶，已被证明在结合(3，4，5)时能切割DNA。我们将对FOK I N末端(FN)催化亚基进行基因工程，使其在其一端与大肠杆菌的生物素化多肽融合。这一序列导致大肠杆菌将其融合到的表达蛋白生物醇化。这种融合的FN蛋白只有在与链霉亲和素结合的复合体中才能切割DNA，链霉亲和素又与上述杂交的生物素结合。这一方案的识别和切割元件的组合将产生“设计的”限制性内切酶，允许在精确选择的位置切割DNA。在第一阶段之前，我们成功地克隆和表达了Fokl(FN)工程蛋白，并证明其具有酶活性。以质粒模板证实了recA介导的启动子插入反应。在第一阶段，我们将改进生物素化FN酶的表达和纯化，并测量recA介导的TON与大片段DNA的结合。将对这两种成分进行测试，以确定它们是否专门切割大DNA。由此产生的产品，作为试剂盒和服务，适用于全球4.5万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.