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Streptavidin homologs for applications on solid surfaces at high temperatures.

One of the most commonly used bonds between two biomolecules is the bond between biotin and streptavidin (SA) or streptavidin homologs (SAHs). A high dissociation constant and the consequent high-temperature stability even allows for its use in nucleic acid detection under PCR conditions. There are a number of SAHs available and for assay design, it is of great interest which SAH will perform best under assay conditions. Albeit 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). We designed multiplex microbead-based experiments and analyzed simultaneously the binding of biotinylated oligonucleotides, 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 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 as 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, 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 even under PCR conditions, regardless of whether they were a Streptavidin variant with slower biotin dissociation and increased mechanostability.