A universal DNA endonuclease

描述（由申请人提供）：本提案的目标是设计具有剪裁切割特异性的通用限制性内切酶。通用内切酶将包含两个功能成分：1)特异性决定因子和2)切割结构域。DNA结合特异性将由修饰的单链寡核苷酸（ss oligo）或肽核酸寡聚物（PNA）贡献。切割结构域来源于IIS型限制性内切酶（REase）或非特异性核酸酶的切割结构域。第一种方法涉及链亲和素和核酸酶切割结构域的融合。嵌合核酸酶，链亲和素核酸酶融合蛋白，通过非共价结合与生物素化的寡核苷酸相连。将在合适的DNA底物上检测嵌合核酸酶的裂解活性。第二种方法将利用中间介导的蛋白质连接（IPL）探索ss寡核苷酸或PNA与核酸酶结构域的共价连接。IPL反应需要半胱氨酸，该半胱氨酸将在化学合成过程中并入低聚物或PNA。对于通用核酸酶的体内应用，寡核苷酸- cys将被修饰为含有锁定核苷酸（LNA）。这种低聚rna能够抵抗内源性内切酶和外切酶的攻击。辅助蛋白如RecA，解旋酶和SSB将被评估以促进目标DNA的结合和切割。对于通用核酸酶在50℃-70℃的等温应用，核酸酶结构域将来自于嗜热性内切酶。

DESCRIPTION (provided by applicant): The goal of this proposal is to engineer universal restriction endonucleases with tailored cleavage specificities. The universal endonucleases will contain two functional components: 1) a specificity determinant and 2) a cleavage domain. The DNA binding specificity will be contributed by a modified singlestranded oligonucleotide (ss oligo) or a peptide nucleic acid oligomer (PNA). The cleavage domain will be derived from the cleavage domain of a type IIS restriction endonuclease (REase) or a non-specific nuclease. The first approach involves fusion of streptavidin and a nuclease cleavage domain. The chimeric nuclease, streptavidin-nuclease fusion protein, is linked to a biotinylated ss oligo through non-covalent binding. The cleavage activity of the chimeric nuclease will be examined on appropriate DNA substrates-. The second approach will explore the covalent attachment of an ss oligo or a PNA to the nuclease domain using inteinmediated protein ligation (IPL). The IPL reaction requires a cysteine which will be incorporated into the oligo or PNA during chemical synthesis. For in vivo applications of the universal nuclease, the oligonucleotide-Cys will be modified to contain locked nucleotides (LNA). Such LNA-oligos are resistant to endogeneous endonuclease and exonuclease attack. Accessory proteins such as RecA, helicase, and SSB will be evaluated to facilitate target DNA binding and cleavage. For isothermal applications of the universal nuclease at 50oC-70oC, the nuclease domain will be derived from thermophilic endonucleases.