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Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry
In This Article
Summary
This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the interaction.
Abstract
Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of various pathways. Key factors such as the structure, sequence, and length of a DNA molecule can significantly influence protein binding. Bio-layer interferometry (BLI) is a label-free technique that measures binding kinetics between molecules, offering a straightforward and precise approach to quantitatively study protein-DNA interactions. A major advantage of BLI over traditional gel-based methods is its ability to provide real-time data on binding kinetics, enabling accurate measurement of the equilibrium dissociation constant (KD) for dynamic protein-DNA interactions. This article presents a basic protocol for determining the KD value of the interaction between a DNA replication protein, replication protein A (RPA), and a single-stranded DNA (ssDNA) substrate. RPA binds ssDNA with high affinity but must also be easily displaced to facilitate subsequent protein interactions within biological pathways. In the described BLI assay, biotinylated ssDNA is immobilized on a streptavidin-coated biosensor. The binding kinetics (association and dissociation) of RPA to the biosensor-bound DNA are then measured. The resulting data are analyzed to derive precise values for the association rate constant (ka), dissociation rate constant (kd), and equilibrium binding constant (KD) using system software.
Introduction
Cellular proteins play a pivotal role in orchestrating the complex biological processes that occur within living organisms. The optimal functioning of these pathways is dependent on the interplay between proteins and other biomolecules inside the cell, including interactions with partner proteins and nucleic acids1. Thus, comprehending the intricacies of cellular processes necessitates a deep understanding of the dynamics of protein-nucleic acid interactions.
Traditionally, protein-nucleic acid interactions have been studied using electrophoretic mobility gel shift assays (EMSAs)2. In this ass....
Protocol
Details of the reagents and the equipment used in the study are listed in the Table of Materials.
1. Preparation of bait, analyte, buffers and drop-holder cleaning
- Setting up the instrument: Turn the instrument on at least 1 h before starting the experiment. This will allow the lamp to warm up.
- Preparation of stripping and cleaning buffers
- Stripping buffer: Prepare 50 mL of 0.15 M Phosphoric acid [pH 2.0]).
- Cleaning.......
Representative Results
In BLI, white light is reflected from the interface of biolayer/buffer and the internal reference interface to the spectrometer. The resulting interference pattern is recorded, and the spectral shift is measured over a period of time and depicted as binding curves response in nm. A representative figure showing the biosensor tip with and without analyte binding and the corresponding spectral wavelength shift (nm) is shown in Figure 1. The binding curves correspond to the baselines, bait load.......
Discussion
The ability to analyze the binding kinetics of any protein to its substrate using BLI provides the means to isolate and characterize the specific factors (such as sequence, structure, or length of a DNA) governing protein-DNA interactions within the cell19. The Octet N1 system, which relies on the principles of biolayer interferometry, allows for quantitative measurement of protein-protein and protein-nucleic acids interactions. Further, interactions between lipids, antibodies, and small molecules.......
Acknowledgements
This work was funded by grants from the National Science Foundation (1929346) and the American Cancer Society (RSG-21-028-01). We would also like to thank members of the Balakrishnan laboratory for helpful discussions.
....Materials
| Name | Company | Catalog Number | Comments |
| 0.5 mL Micro Centrifuge Tubes | Globe Scientific | 111554A | |
| 96 Well Standard Black Microplate | Dot Scientific | 4ti-0223 | |
| Biotinylated poly dT Oligonucleotide | IDT | ||
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A2153-10G | |
| Dithiothreitol (DTT) | Dot Scientific | DSD11000-10 | |
| Ethylenediaminetetraacetic acid (EDTA) | Dot Scientific | DSE57020-500 | |
| Hydrochloric Acid | Fisher Scientific | A144-500 | |
| Kimtec Science Kimwipes | Kimtech | 34120 | |
| Octet N1 Software | Sartorius | 1.4.0.13 | |
| Octet SA Biosensor | Sartorius | 18-5019 | |
| PBS pH 7.2 (10x) | Gibco | 1666711 | |
| Personal Assay Octet N1 System | Sartorius | ||
| Phosphoric Acid | Ward's Science | 470302-024 | |
| Sodium Chloride (NaCl) | Dot Scientific | DSS23020-5000 | |
| Tris Base | Dot Scientific | DST60040-5000 | |
| Tween20 | Bio-Rad | 170-6531 |
References
- Kalodimos, C. G. et al. Structure and flexibility adaptation in non-specific and specific protein-DNA complexes. Science. 305 (5682), 386-389 (2004).
- Fried, M., Crothers, D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9 (23), 6505-6525 (1981).
- Lane, D., Prentki, P., Chandler, M. Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol Rev. 56 (4), 509-528 (1992).
- Dyer, R. B., Herzog, N. K. Immunodepletion EMSA: A novel method to identify proteins in a protein-DNA com....
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