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。

• 批准号: 9317146
• 负责人: VLADIMIR V DIDENKO
• 金额: $ 22.31万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9317146
• 关键词: Acanthamoeba; Aliquot; Alpha Cell; Animals; Apoptosis; Apoptotic; Area; Biological Assay; Biological Markers; Biomedical Research; Caliber; Cell Death; Cell Nucleus; Cells; Characteristics; DNA; DNA Double Strand Break; DNA Ligases; DNA Ligation; DNA Sequence; DNA Topoisomerases; Degenerative Disorder; Detection; Development; Disease; Enzymes; Evolution; Fluorescence; Fluorescence Microscopy; Goals; Heart Diseases; Histologic; Imagery; In Situ; Label; Ligation; Malignant Neoplasms; Modeling; Morphology; Outcome; Pathology; Phosphoric Monoester Hydrolases; Property; Proteins; Reaction; Recombinant Proteins; Recombinants; Research; Sampling; Sensitivity and Specificity; Signal Transduction; Site; Specificity; Speed; Spottings; Stroke; Surface; Techniques; Technology; Testing; Time; Tissues; Topoisomerase; Virus; Work; anticancer research; base; bioimaging; design; fluorescence imaging; histological specimens; in vitro Model; innovation; novel; nuclease; viral DNA

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.

通过磷酸酶辅助拓扑的特异性荧光成像的特异性荧光成像。 抽象的 从Mimivirus获得的DNA序列开始，我们合成了一个异常的病毒DNA拓扑酶。 我们发现，重组蛋白具有极快的连接活性。拓扑异构酶可以 迅速结合DNA结束，在加入DNA后几秒钟内完成了反应。这超过了 所有已知的DNA连接酶的速度通过两个数量级。它允许新颖的超快速和特定 用于可视化组织切片中凋亡细胞的测定法。在这个项目中，我们将开发一个新的表达 测定原位研究。根据检测 由凋亡execution子核能产生的特征性双链DNA断裂。现在 这些生物标志物被认为是对程序性细胞死亡的高度特异性，但它们的检测需要24小时。 拟议的技术将在几分钟之内执行此类检测。现在没有类似的测定 能力。 该项目将采用Mimivirus的独特特性引入一种新的超快速生物成像方法 拓扑异构酶。它将缩小技术差距，并将创建一种有利的标签技术 广泛的应用程序字段。新的快速和特定测定将在生物医学中有用，尤其是 细胞凋亡研究，在对病理样本的明确评估和大量研究的研究中 对程序细胞死亡细胞的定量至关重要，例如癌症，缺血性疾病和 退化性疾病。 该提案的具体目的是： 1）开发第一个明确测定，用于特异性检测组织学中凋亡的特异性检测 凋亡execution骨核能产生的钝的5'PO4 DNA断裂。该测定法将采用 新型磷酸酶辅助拓扑标记技术。使用体外测试新的标签方法 型号。 2）在几种凋亡模型中验证新测定法。优化其速度，灵敏度，特异性和 在固定细胞和组织切片中的适用性。