Engineering DNA nicking endonucleases

描述（由申请人提供）： 在I期，克隆并测序了cviPIIM、cviPIINt、cviQXIM、cviQXINt基因。重组Nt.CviPII在大肠杆菌中表达并纯化。在大肠杆菌中并通过体外转录/翻译系统表达CviQXI。使用新的两步遗传选择程序从Sapl核酸内切酶工程化Nt.Sapl和Nb.Sapl。在II期资助下，我们将优化Nt.CviPII和Nt.CviQXI的表达条件，以减轻细胞毒性。将实现M.CviPII和M.CviQXI的组成型表达以完全修饰宿主DNA。Nt.CviPII和Nt.CviQXI NEases将在pACYC-T7中表达。此外，将构建Nt.CviPII和Nt.CviQXI的C-末端截短变体（与内含肽和几丁质结合结构域融合）。合成肽将通过内含肽介导的肽连接与截短形式连接以构建全长酶。将二级氨基酸（aa）取代引入Nb.Sapl中以优化切口活性并消除dsDNA切割。将等效aa变化引入BspQI（一种热稳定Sapl异构体）中，以分离N. BspQI。Sapl和Earl共享相似的DNA识别序列，并且两种核酸内切酶的aa序列显示出高度的相似性。将导致SapI切口变体的aa变化引入Earl内切核酸酶中以分离N. Earl。我们将从4-碱基切割子MnII（CCTCN 7 A，ANeGAGG）分离切口变体。还将从在75 ℃下有活性的Tth 111 II（CAARCA 11/9）中分离出极端热稳定的切口变体。我们将从归巢核酸内切酶l-Scel和l-Dmol设计具有15 -18 bp识别序列的切口变体。我们将克隆和表征HNH型归巢核酸内切酶从克氏杆菌病毒基因组。在第一阶段，我们发现了一种新的DNA扩增方法，使用Nt.CviPII和Bst DNA聚合酶。已经从少量基因组DNA或从单个细菌菌落实现了随机DNA扩增，而无需添加外源引物。在第二阶段的资助下，这种等温扩增方法（NEMDA）将得到优化，灵敏度将得到提高。最后，我们将研究NEMDA检测病原体的潜力及其在生物勘探中的应用。

DESCRIPTION (provided by applicant): In Phase I, the cviPIIM, cviPIINt, cviQXIM, cviQXINt genes were cloned and sequenced. Recombinant Nt.CviPII was expressed in E.coli and purified. Nt.CviQXI was expressed in E.coli and by an in vitro transcription/translation system. Nt.Sapl and Nb.Sapl were engineered from Sapl endonuclease using a novel two-step genetic selection procedure. Under Phase II funding, we will optimize the expression conditions for Nt.CviPII and Nt.CviQXI to alleviate cytotoxicity. Constitutive expression of M.CviPII and M.CviQXI will be achieved to fully modify host DNA. Nt.CviPII and Nt.CviQXI NEases will be expressed in pACYC-T7. In addition, C-terminal truncation variants of Nt.CviPII and Nt.CviQXI will be constructed (in fusion with intein and chitin binding domain). Synthetic peptides will be ligated to the truncated versions via intein-mediated peptide ligation to construct full-length enzymes. Secondary amino acid (aa) substitutions will be introduced into Nb.Sapl to optimize the nicking activity and eliminate dsDNA cleavage. The equivalent aa changes will be introduced into BspQI (a thermostable Sapl isoschizomer) to isolate N.BspQI. Sapl and Earl share similar DNA recognition sequences and the aa sequences of the two endonucleases display a high level of similarity. The aa changes that resulted in Sapl nicking variants will be introduced into Earl endonuclease to isolate N.Earl. We will isolate nicking variants from a 4-base cutter Mnll (CCTCN7 A, ANeGAGG). Extremely thermostable nicking variants will also be isolated from Tth111II (CAARCA11/9) that is active at 75 degrees C. We will engineer nicking variants from homing endonucleases l-Scel and l-Dmol with15-18 bp recognition sequences. We will clone and characterize HNH type homing endonucleases from chlorella virus genomes. In Phase I, we discovered a novel DNA amplification method using Nt.CviPII and Bst DNA polymerase. Random DNA amplification has been achieved from a small amount of genomic DNA or from a single bacterial colony without addition of exogenous primers. Under Phase II funding, this isothermal amplification method (NEMDA) will be optimized and the sensitivity will be improved. Finally, we will investigate the potential of NEMDA for detections of pathogens and its use in bio-prospecting.