Abstract
Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots) have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios. So far, however, there is no convenient way to label proteins purified from budding yeast with Qdots. Based on BirA–Avi and biotin–streptavidin systems, we have established a simple method to acquire a Qdot-labeled protein and visualize its interaction with DNA using total internal reflection fluorescence microscopy. For proof-of-concept, we chose replication protein A (RPA) and origin recognition complex (ORC) as the proteins of interest. Proteins were purified from budding yeast with high biotinylation efficiency and rapidly labeled with streptavidin-coated Qdots. Interactions between proteins and DNA were observed successfully at the single-molecule level.
Highlights
Understanding the dynamic complexity of life process is one of the major goals in molecular biology
The biotin carboxyl carrier protein (BCCP) subunit of acetylCoA carboxylase can be biotinylated by the biotin holoenzyme synthetase, BirA, at the epsilon amino group of a specific lysine residue in E. coli
To acquire the biotinylated proteins in budding yeast, in this study, we combined the BirA–Avi and GAL1GAL10 promoter-driven expression systems to establish a convenient method for obtaining biotinylated target proteins in budding yeast
Summary
Understanding the dynamic complexity of life process is one of the major goals in molecular biology. The rapidly developing technique of single-molecule fluorescence microscopy has made great contributions to revealing the details of nucleic acid and protein interactions in various biological processes, such as DNA replication (Yardimci et al, 2012; Duzdevich et al, 2015; Ticau et al, 2015; Graham et al, 2017), spliceosome (Fareh et al, 2016), and CRISPR-Cas systems (Redding et al, 2015). Compared with organic dyes and fluorescent proteins, Qdots have several unique merits, including intense brightness, exceptional photostability, and large Stokes shift. They are suitable for long-time observation and enable signal-to-noise ratio improvement (Medintz et al, 2005; Nelson et al, 2011)
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