Engineering DNA nicking endonucleases

工程 DNA 切口核酸内切酶

• 批准号: 7119677
• 负责人: SHUANG-YONG XU
• 金额: $ 28.42万
• 依托单位: NEW ENGLAND BIOLABS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-05 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7119677
• 关键词: Escherichia coli; endonuclease; enzyme mechanism; enzyme model; gel mobility shift assay; genetic polymorphism; nucleic acid amplification techniques; nucleic acid hybridization; nucleic acid sequence; nucleic acid structure; protein engineering; technology /technique development; thermostability

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.

描述(由申请人提供)： 在第一阶段，克隆了cviPIIM、cviPIINt、cviQXIM、cviQXINt基因。重组Nt.CviPII在大肠杆菌中表达并纯化。Nt.CviQXI在大肠杆菌中表达，并通过体外转录/翻译系统进行表达。Nt.Sap1和Nb.Sap1是通过一种新的两步遗传选择程序从Sap1内切酶中获得的。在第二阶段的资助下，我们将优化Nt.CviPII和Nt.CviQXI的表达条件，以减轻细胞毒性。实现M.CviPII和M.CviQXI的组成性表达，以充分修饰宿主DNA。Nt.CviPII和Nt.CviQXI NEase将在pACYC-T7中表达。此外，还将构建Nt.CviPII和Nt.CviQXI的C-末端截断变体(与内含素和几丁质结合域融合)。合成肽将通过内含素介导的肽连接连接到截短的版本上，以构建全长酶。Nb.Sapl将引入二次氨基酸(AA)取代，以优化其划痕活性并消除dsDNA切割。等量的AA变化将引入BspQI(一种耐热的Sapl异构体)以分离N.BspQI。SAPL和Earl具有相似的DNA识别序列，两种内切酶的AA序列显示出很高的相似性。导致Sapl划痕变异的AA变化将被引入Earl内切酶中，以分离N.Earl。我们将从4碱基刀具Mnll(CCTCN7 A，ANeGAGG)中分离出划痕变体。还将从75℃下活跃的Tth111II(CAARCA11/9)中分离出极耐热的划痕变异体。我们将从归巢内切酶L-SCEL和L-Dmol中设计具有15-18个核苷酸识别序列的划痕变异体。我们将从小球藻病毒基因组中克隆和鉴定HNH型归巢内切酶。在第一阶段，我们发现了一种利用Nt.CviPII和BST DNA聚合酶进行DNA扩增的新方法。从少量的基因组DNA或从单个细菌菌落中进行随机DNA扩增，无需添加外源引物。在第二阶段的资助下，这种等温放大方法(NEMDA)将得到优化，灵敏度将得到提高。最后，我们将研究NEMDA在病原体检测方面的潜力及其在生物勘探中的应用。

项目成果

Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI.

• DOI: 10.1093/nar/gkm481
• 发表时间: 2007
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Xu, Shuang-Yong;Zhu, Zhenyu;Zhang, Penghua;Chan, Siu-Hong;Samuelson, James C;Xiao, Jianping;Ingalls, Debra;Wilson, Geoffrey G
• 通讯作者: Wilson, Geoffrey G