Investigation of molecular beacon aptamer-based bioassay for platelet-derived growth factor detection
Investigation of molecular beacon aptamer-based bioassay for platelet-derived growth factor detection
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DOI：
10.1002/cbic.200400308
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发表时间：
2005-05-01
期刊：
影响因子：
3.2
通讯作者：
Tan, WH
中科院分区：
文献类型：
--
作者：
Vicens, MC;Sen, A;Tan, WH
本报告介绍了使用分子信标探针(MBA)作为合成的高亲和力DNA探针的研究，该探针对特定的蛋白质生物标记物--血小板衍生生长因子(PDGF)产生荧光共振能量转移(FRET)。作为将MBA应用于基于荧光的生物标本分析的一步，我们检查了孵育的某些物理和化学参数的影响，这些参数将影响DNA构象和DNA骨架修饰，从而提高核糖抗性。这种生物测定与生物样品中通常遇到的pH、温度和单价阳离子水平兼容，并且硫代骨架修饰的MBA能够在最少的样品处理和没有测试优化的情况下显示出特定的FRET，MBA能够从细胞培养液中检测到低至每微克血清蛋白10 ng的PDGF。我们还表明，不同的已知荧光团-猝灭剂对可以成功地用于MBA，以灵敏地检测PDGF靶标。因此，应该有可能开发多种生物检测方法，通过使用由所需蛋白质靶标的高亲和力DNA配体创建的MBA来监控猝灭或增强以同时检测几个生物标记物。有趣的是，我们观察到，对于标准多聚体蛋白靶标具有多个结合位点的DNA配体，FRET生物测定可以通过使用两个单独标记的DNA的混合物来完成-一个携带荧光团，另一个带有匹配的猝灭剂。这一观察结果对未来设计更具选择性的基于DNA的FRET生物检测方法具有重要意义，该方法针对某些蛋白质靶标使用不止一个配体。
This report describes studies on the use of a molecular-beacon aptomer (MBA) as a synthetic high-affinity DNA probe that exhibits fluorescence resonance energy transfer (FRET) in response to a specific protein biomarker, platelet-derived growth factor (PDGF). As a step toward the application of the MBA in a fluorescence-based assay for biological specimens, we examined the influence of certain physical and chemical parameters of incubation that would affect DNA conformation and DNA-backbone modification, and thus improve nucleose resistance. This bioassay is compatible with pH, temperature, and monovolent cation levels typically encountered in biological samples, and phosphorothioate backbone-modified MBA is able to exhibit specific FRET With minimal sample processing and without assay optimization, the MBA is able to detect as little as 10 ng PDGF per mu g of serum proteins from cell-culture media. We also show that different sets of known fluorophore-quencher pairs can be successfully used in the MBA for sensitive detection of the PDGF target. It should, therefore, be possible to develop multiplex bioassays that monitor either quenching or enhancement for the simultaneous detection of several biomarkers by using MBAs created from high-affinity DNA ligands for the desired protein targets. Interestingly, we observed that, with a DNA ligand with multiple binding sites for a standard multimeric protein target, the FRET bioassay could be accomplished by using a mixture of two individually labeled DNAs-one carrying the fluorophore and the other with the matching quencher. This observation has significant implications in the future design of more selective DNA-based FRET bioassays that use more than one ligand for the some protein target.