Immobilization of Single Biomolecules Using Covalent-Bond Linkages for Fluorescence Single-Molecule Experiments

Abstract

The streptavidin-biotin bridge is commonly used in single-molecule studies to surface immobilize biomolecules onto microscope slides. However, the presence of tryptophanes impedes utilization of UV light and numerous fluorescent nucleotide analogs, such as 2-aminopurine. We are developing new approaches to immobilize DNA/RNA molecules without use of streptavidin and biotin. One approach consists of using the Huisgen cycloaddition reaction between an alkyne and an azide, which is an example of “click” chemistry reaction. In this “click” chemistry approach, 3′-azide modified oligos are immobilized to an alkyne-modified microscope slide surface through a triazole linkage. This cycloaddition reaction is very stable in many physiologically relevant buffers, and has been shown to occur without the need of a catalyst. In another approach, we take advantage of the efficient coupling between thiol groups to immobilize biomolecules by forming disulfide bridges. 3′-thiol modified oligos are surface immobilized on a thiol-modified microscope slide by forming disulfide bonds. We are currently improving the immobilization efficiency by optimizing the reaction parameters and conditions. We anticipate that these approaches will allow us to investigate local conformational changes in biomolecular systems at the single molecule level.