Faculty of Environment and Natural Sciences, Institute of Biotechnology , Brandenburg University of Technology Cottbus-Senftenberg , Universitätsplatz 1 , D-01968 Senftenberg , Germany.
Optical Spectroscopy and Chemical Imaging, Institute of Chemistry , University of Potsdam , Karl-Liebknecht-Straße 24-25, Building 29 , D-14476 Potsdam , Germany.
Langmuir. 2020 Jan 21;36(2):628-636. doi: 10.1021/acs.langmuir.9b02339. Epub 2020 Jan 10.
One of the most commonly used bonds between two biomolecules is the bond between biotin and streptavidin (SA) or streptavidin homologues (SAHs). A high dissociation constant and the consequent high-temperature stability even allows for its use in nucleic acid detection under polymerase chain reaction (PCR) conditions. There are a number of SAHs available, and for assay design, it is of great interest to determine as to which SAH will perform the best under assay conditions. Although there are numerous single studies on the characterization of SAHs in solution or selected solid phases, there is no systematic study comparing different SAHs for biomolecule-binding, hybridization, and PCR assays on solid phases. We compared streptavidin, core streptavidin, traptavidin, core traptavidin, neutravidin, and monomeric streptavidin on the surface of microbeads (10-15 μm in diameter) and designed multiplex microbead-based experiments and analyzed simultaneously the binding of biotinylated oligonucleotides and the hybridization of oligonucleotides to complementary capture probes. We also bound comparably large DNA origamis to capture probes on the microbead surface. We used a real-time fluorescence microscopy imaging platform, with which it is possible to subject samples to a programmable time and temperature profile and to record binding processes on the microbead surface depending on the time and temperature. With the exception of core traptavidin and monomeric streptavidin, all other SA/SAHs were suitable for our investigations. We found hybridization efficiencies close to 100% for streptavidin, core streptavidin, traptavidin, and neutravidin. These could all be considered equally suitable for hybridization, PCR applications, and melting point analysis. The SA/SAH-biotin bond was temperature-sensitive when the oligonucleotide was mono-biotinylated, with traptavidin being the most stable followed by streptavidin and neutravidin. Mono-biotinylated oligonucleotides can be used in experiments with temperatures up to 70 °C. When oligonucleotides were bis-biotinylated, all SA/SAH-biotin bonds had similar temperature stability under PCR conditions, even if they comprised a streptavidin variant with slower biotin dissociation and increased mechanostability.
生物分子之间最常用的键之一是生物素与链霉亲和素（SA）或链霉亲和素类似物（SAH）之间的键。高离解常数和随之而来的高温稳定性甚至允许其在聚合酶链反应（PCR）条件下用于核酸检测。有许多 SAH 可用，对于测定设计，非常感兴趣的是确定在测定条件下哪种 SAH 表现最佳。尽管有许多关于 SAH 在溶液或选定固相中的特性的单一研究，但没有系统的研究比较不同的 SAH 用于固相中的生物分子结合、杂交和 PCR 测定。我们比较了链霉亲和素、核心链霉亲和素、traptavidin、核心 traptavidin、中性亲和素和单体链霉亲和素在微珠（直径 10-15μm）表面上的性能，并设计了多重微珠实验，同时分析了生物素化寡核苷酸的结合和寡核苷酸与互补捕获探针的杂交。我们还将类似大的 DNA 折纸与微珠表面上的捕获探针结合。我们使用实时荧光显微镜成像平台，该平台可以使样品经历可编程的时间和温度曲线，并根据时间和温度记录微珠表面上的结合过程。除了核心 traptavidin 和单体链霉亲和素外，所有其他 SA/SAH 都适合我们的研究。我们发现链霉亲和素、核心链霉亲和素、traptavidin 和中性亲和素的杂交效率接近 100%。这些都可以被认为同样适合杂交、PCR 应用和熔点分析。当寡核苷酸单生物素化时，SA/SAH-生物素键对温度敏感，其中 traptavidin 最稳定，其次是链霉亲和素和中性亲和素。单生物素化的寡核苷酸可用于温度高达 70°C 的实验。当寡核苷酸双生物素化时，所有 SA/SAH-生物素键在 PCR 条件下具有相似的温度稳定性，即使它们包含具有较慢生物素解离和增加机械稳定性的链霉亲和素变体也是如此。
