DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level
Abstract
:1. Introduction
2. Main Single-Molecule Techniques for Studying Protein-DNA Interactions
3. DNA Flow-Stretch Assays
3.1. Traditional Approach
3.2. Magnetic Bead-Based Approach
3.3. DNA Tightropes
4. Traditional DNA Curtains
4.1. Technological Design
4.2. Single-Tethered DNA Curtains
4.3. Double-Tethered DNA Curtains
4.4. Other Configurations
4.5. Single-Stranded DNA Curtains
4.6. Combination with Other Techniques
5. Other Types of DNA Curtains
6. Future Perspectives
Author Contributions
Funding
Conflicts of Interest
References
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Technique | Technological Basis | Benefits | Limitations | Key References |
---|---|---|---|---|
Traditional biochemical methods (electrophoretic mobility shift assay, cross-linking, footprinting, chromatin immunoprecipitation and others) | Depends on a specific method. | Enables identification of protein-DNA complexes, discovery of protein DNA-binding sites localization, quantification of the kinetics and affinity of protein-DNA interactions and can predict the location of a specific DNA-binding protein on a genome-wide scale. | Based solely on ensemble measurements, thus hiding the real-time dynamics and unique features of individual protein-DNA complexes. Provides only the averaged characteristics of such complexes. | [10,12] |
Tethered particle motion | One end of the DNA molecule is immobilized on the surface, while the other end is attached to a metallic or polystyrene bead. | Monitors DNA length changes. Particularly suitable for studying proteins that induce a shortening of the DNA molecule. | Low temporal resolution (slightly lower than 1 s). Protein-DNA interactions with rapid dynamics are typically unobservable. | [18,20,21] |
Optical tweezers | One (or both) ends of the DNA molecule are tethered to a dielectric bead trapped by a highly focused laser beam, while the other end is anchored on the surface. | Can apply variable pN forces to individual nucleoprotein complexes. Suitable for investigating mechanical properties of DNA and force-dependent kinetic reaction rates of protein-DNA interactions. | Usually allows the manipulation of only one protein-DNA complex at a time. | [22,23] |
Magnetic tweezers | One DNA molecule end is immobilized on the surface, as the second end is attached to a paramagnetic bead. | Confers adjustable pN loads to the DNA molecule by stretching it accordingly. Can induce torque on the DNA allowing one to control its tension. Particularly suitable for probing DNA topology and DNA-supercoiling proteins. | [25,27] | |
Atomic force microscopy (AFM) | Atomically sharp tip of the AFM cantilever scans the sample by interacting with its surface under the action of attractive and repulsive forces. | Directly visualizes individual nucleoprotein complexes at nanometer resolution. Provides structural and stoichiometric information about protein-DNA complexes. Can be modified to observe the formation of such complexes in real time. | Low throughput. Slow scanning speed resulting in thermal drift of the imaged sample. Possible non-specific interaction between AFM probe and the surface of a biological sample. | [22,29,31] |
Single-molecule Förster resonance energy transfer (smFRET) | FRET pair is deployed on a single DNA molecule, or the donor fluorophore is incorporated into a DNA-binding protein, while the acceptor is attached to the DNA. | Enables the observation of DNA-binding protein conformational changes and single protein-DNA-interaction events at the resolution of 1 to 10 nm. | Requires labeling of biomolecules of interest with specific fluorophores. Can only detect conformational changes or protein-binding events in a relatively narrow range. | [23,33,35,36] |
Conventional DNA flow-stretch assays | DNA molecules are immobilized on the surface typically by one of their ends and then stretched along the surface by applying a buffer flow. | Directly visualizes individual protein-DNA interactions and protein translocation along DNA in real time. Characterizes protein-binding profiles and dwell times. | Only tens of individual nucleoprotein complexes can be observed in a single field of view of the microscope. Possible overlapping of surface-immobilized and flow-stretched DNA molecules. Degree of DNA extension and DNA orientation, in terms of a nucleotide sequence, is unknown. | [40,42,56] |
Magnetic bead-based DNA flow-stretch assays | One end of the DNA is tethered to the surface, while the other end is attached to a paramagnetic bead. The latter is subjected to a magnetic force and the DNA molecule is then stretched along the surface by using a hydrodynamic flow. | Directly visualizes individual protein-DNA interactions and protein translocation along DNA in real time. Characterizes protein-binding profiles and dwell times. Suitable for studying more diverse processes such as protein-induced DNA compaction and others. | [77,82,83] | |
DNA tightropes | Both ends of the DNA are anchored on the top of two neighboring micron-sized beads by the means of a buffer flow. | Directly visualizes individual protein-DNA interactions and protein translocation along DNA in real time. Characterizes protein-binding profiles and dwell times. Continuous buffer flow is not required for imaging. Nucleoprotein complexes are located further away from the surface. | [89,90] | |
Traditional DNA curtains | DNA molecules are immobilized on a supported lipid bilayer (SLB) and then stretched and aligned in a parallel manner along the lipid diffusion barriers by employing a hydrodynamic flow. | Directly visualizes individual protein-DNA interactions and protein translocation along DNA in real time. Characterizes protein-binding profiles and dwell times. Enables high-throughput imaging. Extension and orientation of DNA are defined. Can be assembled in different configurations with various DNA substrates. | Fabrication of lipid diffusion barriers is technically challenging and requires specific knowledge and expensive equipment. Possible system stability and defect management issues related to preparation of SLBs. | [50,100,101,102,110,130,145] |
Suspended DNA curtains | DNA molecules are tethered to a microfluidic channel- bisecting gold nanowire and then stretched by applying a buffer flow which determines the parallel arrangement of DNA. | Directly visualizes individual protein-DNA interactions and protein translocation along DNA in real time. Characterizes protein-binding profiles and dwell times. Nucleoprotein complexes are located further away from the surface. | Low-throughput nature of this approach. Substantial overlapping of discrete DNA molecules suspended on nanowire. | [169] |
Soft DNA curtains | DNA molecules are anchored to the protein line-features nanopatterned on the surface of a glass coverslip and then aligned in a parallel manner by a hydrodynamic-flow-induced stretching. | Directly visualizes individual protein-DNA interactions and protein translocation along DNA in real time. Characterizes protein-binding profiles and dwell times. Enables high-throughput imaging. Extension and orientation of DNA are defined. Can be assembled in different configurations. Cost-effective and user-friendly platform. | Considerable non-specific adsorption of DNA-binding proteins on the protein-nanopatterned glass coverslip surface. | [170,171] |
DNA skybridge | Both ends of the DNA are immobilized one after another on adjacent 4 µm high thin quartz barriers by using a buffer flow. | Directly visualizes individual protein-DNA interactions and protein translocation along DNA in real time. Characterizes protein-binding profiles and dwell times. Nucleoprotein complexes are located further away from the surface. Ensures higher signal-to-noise ratios during imaging. | Fabrication of a special 3D structure required for the assembly of DNA skybridge is technically challenging and requires specialized equipment and knowledge. | [172] |
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Kopūstas, A.; Zaremba, M.; Tutkus, M. DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level. Appl. Nano 2022, 3, 16-41. https://doi.org/10.3390/applnano3010002
Kopūstas A, Zaremba M, Tutkus M. DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level. Applied Nano. 2022; 3(1):16-41. https://doi.org/10.3390/applnano3010002
Chicago/Turabian StyleKopūstas, Aurimas, Mindaugas Zaremba, and Marijonas Tutkus. 2022. "DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level" Applied Nano 3, no. 1: 16-41. https://doi.org/10.3390/applnano3010002
APA StyleKopūstas, A., Zaremba, M., & Tutkus, M. (2022). DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level. Applied Nano, 3(1), 16-41. https://doi.org/10.3390/applnano3010002