Express assay for specific fluorescence imaging of apoptosis via phosphatase-assisted topoligation.

磷酸酶辅助拓扑寡核苷酸诱导的细胞凋亡特异性荧光成像的表达分析。 摘要 从从MimiVirus获得的DNA序列出发，我们合成了一种不寻常的病毒DNA拓扑异构酶。 我们发现重组蛋白具有极快的连接活性。拓扑异构酶可以 快速连接DNA结束，在加入DNA后几秒钟内完成反应。这超过了 所有已知的DNA连接酶的速度提高了两个数量级。它允许一种新颖的超快和特定的 组织切片中凋亡细胞的显影分析。在这个项目中，我们将开发一种新的快递 用于现场研究的化验。该检测将根据检测结果选择性地标记凋亡细胞，而不是坏死细胞 由凋亡的执行者核酸酶产生的特有的双链DNA断裂。目前 这些生物标记物被认为是细胞程序性死亡的高度特异性，但它们的检测需要24小时。 这项拟议的技术将在几分钟内完成这种检测。现在还没有类似的检测方法 能力。 该项目将引入一种新的超高速生物成像方法，利用MimiVirus的独特特性 拓扑异构酶。它将缩小技术差距，并将创造一种具有优势的标签技术 应用领域广。这种新的快速和特异的分析方法将在生物医学中有用，特别是在 细胞凋亡研究，在病理样本的快速评估中和在大容量研究中 程序性细胞死亡细胞的数量是必不可少的，例如在癌症、缺血性疾病和 退行性疾病。 这项建议的具体目标是： 1)建立了第一种通过标记细胞凋亡来特异性检测组织切片中的细胞凋亡的表达方法。 由凋亡的执行者核酸酶产生的钝端5‘PO4 DNA断裂。该化验将采用 磷酸酶辅助拓扑配基标记新技术。使用体外实验测试新的标记方法 模特们。 2)在多种细胞凋亡模型中验证新的检测方法。以优化其速度、灵敏度、特异度和 适用于固定细胞和组织切片。

Express assay for specific fluorescence imaging of apoptosis via phosphatase-assisted topoligation. Abstract Starting with DNA sequence obtained from Mimivirus we synthesized an unusual viral DNA topoisomerase. We found that the recombinant protein possesses extremely fast ligation activity. The topoisomerase can rapidly ligate DNA ends, finishing the reaction within several seconds after addition to DNA. This surpasses the speed of all known DNA ligases by two orders of magnitude. It permits a novel ultra-fast and specific assay for visualization of apoptotic cells in tissue sections. In this project we will develop a new express assay for in situ research. The assay will selectively label apoptotic, but not necrotic cells, based on detection of characteristic double-stranded DNA breaks produced by apoptotic executioner nucleases. At present these biomarkers are considered highly specific for programmed cell death, but their detection takes 24 hrs. The proposed technology will perform such detection within minutes. Now there are no assays with similar capability. The project will introduce a new ultra-fast bioimaging approach employing the unique properties of Mimivirus topoisomerase. It will close the technological gap and will create an advantageous labeling technique with a wide application field. The new quick and specific assay will be useful in biomedicine, particularly in apoptosis research, in express assessment of pathology samples and in studies where large-volume quantitations of programmed cell death cells are essential, such as in cancers, ischemic disorders, and degenerative diseases. The Specific Aims of the proposal are: 1) To develop the first express assay for specific detection of apoptosis in histological sections via labeling of blunt-ended 5’PO4 DNA breaks produced by apoptotic executioner nucleases. The assay will employ the novel phosphatase-assisted topoligation labeling technology. To test the new labeling approach using in vitro models. 2) To verify the new assay in several apoptotic models. To optimize its speed, sensitivity, specificity and applicability in fixed cells and tissue sections.