Nucleoprotein assemblies for cellular biomarker detection
Nucleoprotein assemblies for cellular biomarker detection
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DOI：
10.1021/nl060549h
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发表时间：
2006-06-01
期刊：
影响因子：
10.8
通讯作者：
Smith, Steven S.
中科院分区：
文献类型：
--
作者：
Singer, Elizabeth M.;Smith, Steven S.
在本报告中，我们使用 DNA Y 接头作为 EcoRII 甲基转移酶-硫氧还蛋白 (M(.)EcoRII-Trx) 融合蛋白的荧光支架。 DNA 支架和甲基转移酶之间的共价连接在含有 5FdC 的支架上的预选位点形成。发现所得到的硫氧还蛋白靶向纳米装置选择性地与某些细胞系结合，但不与其他细胞系结合。构建融合蛋白以允许硫氧还蛋白肽从纳米装置上蛋白水解裂解。用凝血酶或肠激酶进行蛋白水解，有效地从纳米装置中去除硫氧还蛋白肽，并消除通过荧光测量的细胞系特异性结合。可以预见这种类型的设备有许多潜在的应用。特别是，融合蛋白能够选择性地将纳米装置靶向某些肿瘤细胞系而不是其他细胞系，这表明这种方法可以作为肿瘤分类以及探测细胞表面受体结构和功能中免疫组织化学方法的辅助手段。
In this report, we have used DNA Y-junctions as fluorescent scaffolds for EcoRII methyltransferase-thioredoxin (M(.)EcoRII-Trx) fusion proteins. Covalent links between the DNA scaffold and the methyltransferase were formed at preselected sites on the scaffold containing 5FdC. The resulting thioredoxin-targeted nanodevice was found to bind selectively to certain cell lines but not to others. The fusion protein was constructed so as to permit proteolytic cleavage of the thioredoxin peptide from the nanodevice. Proteolysis with thrombin or enterokinase effectively removed the thioredoxin peptide from the nanodevice and extinguished cell line specific binding measured by fluorescence. A number of potential applications for devices of this type can be envisioned. In particular, the ability of the fused protein to selectively target the nanodevice to certain tumor cell lines and not others suggests that this approach may serve as an adjunct to immunohistochemical methods in tumor classification as well as probe cell surface receptor architecture and function.