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

通过磷酸酶辅助拓扑调节对细胞凋亡进行特异性荧光成像的快速检测。 摘要 从拟菌病毒获得的 DNA 序列开始，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 末端，添加 DNA 后数秒内完成反应。这超越了 所有已知 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速和特定的 组织切片中凋亡细胞可视化的测定。在这个项目中我们将开发一种新的快递 进行原位研究测定。该测定将根据检测选择性标记凋亡细胞，但不标记坏死细胞 凋亡刽子手核酸酶产生的特征性双链 DNA 断裂。目前 这些生物标志物被认为对程序性细胞死亡具有高度特异性，但它们的检测需要 24 小时。 所提出的技术将在几分钟内完成此类检测。目前还没有类似的检测 能力。 该项目将引入一种新的超快速生物成像方法，利用拟菌病毒的独特特性 拓扑异构酶。它将缩小技术差距，并创造一种具有优势的标签技术 应用领域广泛。新的快速且特异性的测定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和大容量研究中 程序性细胞死亡细胞的定量至关重要，例如在癌症、缺血性疾病和 退行性疾病。 该提案的具体目标是： 1) 开发第一个通过标记来特异性检测组织学切片中细胞凋亡的快速检测方法 由凋亡刽子手核酸酶产生的平端 5'PO4 DNA 断裂。该测定将采用 新型磷酸酶辅助拓扑标记技术。使用体外测试新的标记方法 模型。 2) 在几种细胞凋亡模型中验证新的检测方法。优化其速度、灵敏度、特异性和 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.