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Oriented Soft DNA Curtains for Single-Molecule Imaging
Aurimas Kopu Stas1, Šaru Nė Ivanovaitė, Tomas Rakickas
1Life Sciences Center, Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.
Langmuir : the ACS Journal of Surfaces and Colloids
|March 10, 2021
Summary
New DNA Curtains use protein templates for stable, oriented DNA stretching, enabling precise study of protein-DNA interactions like CRISPR-Cas9 binding. This advancement enhances biophysical studies with faster data acquisition and controlled conditions.
Area of Science:
- Biophysics
- Nanotechnology
- Molecular Biology
Background:
- Single-molecule methods are crucial for biophysical studies.
- Nanotechnology platforms enhance experimental design and data acquisition speed.
- DNA Curtains are a key nanotechnological platform for DNA stretching.
Purpose of the Study:
- To develop advanced strategies for fabricating stable, oriented DNA molecule arrays using protein template-directed assembly.
- To improve DNA molecule immobilization and extension for controlled biophysical experiments.
- To demonstrate the utility of the improved DNA Curtains for studying protein-DNA interactions.
Main Methods:
- Fabrication of protein templates on glass coverslips for directional DNA assembly.
- Utilizing biotinylated DNA molecules for immobilization onto protein templates.
- Employing single-end and both-end immobilization strategies, including heterologous DNA labeling and antibody use.
- Using traptavidin for enhanced immobilization stability.
- Demonstrating the assay with fluorescently labeled nucleic acid-interacting proteins, such as CRISPR-Cas9.
Main Results:
- Successfully created oriented arrays of stably immobilized DNA molecules using protein templates.
- Developed a both-end immobilization method that eliminates the need for continuous buffer flow, allowing for more controlled reaction conditions.
- Achieved enhanced immobilization stability of DNA molecules.
- Validated the double-tethered Soft DNA Curtains for monitoring protein binding location and position on individual DNA molecules.
Conclusions:
- The developed protein template-directed assembly offers a robust method for creating stable, oriented DNA Curtains.
- Both-end immobilization provides superior control over reaction conditions for studying protein-DNA interactions.
- This improved DNA Curtains platform is effective for high-resolution analysis of nucleic acid-binding proteins, including CRISPR-Cas9.
