Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA–Protein Interactions at the Single-Molecule Level

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.

Structural insights into multifunctionality of human FACT complex subunit hSSRP1

“…To obtain information about the binding kinetics of hSSRP1-HMGD to DNA, biotin-labeled lambda-phage DNA molecules attached to beads were held by an optical trap and extended by flowing the buffer in a two-channel flow cell, as previously described ( 72 , 73 ). The binding buffer used in these experiments contained 10 mM Tris, 20 mM EDTA, 25 mM KCl, 0.2 mg/l bovine serum albumin, and 10 mM MgCl 2 .…”

Bio-SCAN: A CRISPR/dCas9-Based Lateral Flow Assay for Rapid, Specific, and Sensitive Detection of SARS-CoV-2

“… 45 The AviTag is currently used for protein purification, to trap and purify interacting proteins, and to determine protein–DNA interactions. 14 , 16 , 43 , 46 …”

“…Lateral flow assays (LFAs) are simple diagnostic methods for pathogen detection in nonlaboratory settings, as they are rapid, cost-effective, and user-friendly. , The first commercial application of LFAs, almost 50 years ago, was in pregnancy tests to detect human chorionic gonadotropin in urine; since then, LFAs have become one of the most reliable tools to detect disease markers for medical diagnostics. − The strong, noncovalent interaction between biotin and avidin is widely used to purify proteins and nucleic acids or isolate interacting partners. − Therefore, LFA strips were developed to detect proteins or nucleic acids by incorporating the biotin–avidin interaction. , Biotin–avidin-based LFAs have recently emerged as a convenient approach for the detection of pathogen-derived nucleic acids . Strips are impregnated with avidin/streptavidin at the test line (test line, T) that specifically immobilizes biotin-labeled molecules present in the lateral flow, followed by visual detection. , However, direct use of biotin–streptavidin-based LFAs suffer from low sensitivity, nonspecific interactions, false detection, and the inability to differentiate correct amplicons from primer dimers.…”

“…In molecular biology, biotin labeling, that is, proximity-dependent biotin identification (BioID and TurboID), is a well-established method to test protein–protein interactions or identify interacting partners in living cells. ,, Biotin can be added to any protein by chemically modifying primary amino groups (−NH 2 ) or enzymatic labeling with biotin, this latter method being generally preferred due to the resulting homogeneity and ease of labeling. , In Escherichia coli, biotin ligase (BirA) specifically attaches biotin to a 75-residue fragment of biotin carboxyl carrier protein (BCCP), which was later optimized to a 14-amino acid sequence (AviTag) as the substrate for BirA . The AviTag is currently used for protein purification, to trap and purify interacting proteins, and to determine protein–DNA interactions. ,,, …”