研究目的
Investigating the control of diffusional behavior and assembly of DNA nanostructures on lipid membranes using ion concentration changes and super-resolution imaging.
研究成果
The study demonstrates a reversible method to control the diffusion and assembly of DNA nanostructures on lipid membranes using ion concentration changes, enabling super-resolution imaging of dynamic processes. This approach enhances assembly yields in 2D and provides insights into membrane-associated behaviors, with potential applications in studying biological mechanisms and controlling membrane cargos.
研究不足
The molecular mechanism for transient immobilization is unclear. The method may not work on all substrates (e.g., mica-supported SLBs show residual movement). Sample preparation requires specific conditions, such as KOH treatment for glass surfaces to reduce roughness, and assembly yields can be low without purification.
1：Experimental Design and Method Selection:
The study uses DNA origami structures designed with CaDNAno 2.0, folded and purified, and attached to supported lipid bilayers (SLBs) via cholesterol-modified oligonucleotides. Diffusion is measured using single-particle tracking with a custom-built TIRF microscope, and super-resolution imaging is performed with DNA-PAINT microscopy. Salt concentrations (NaCl and MgCl2) are varied to control diffusion and immobilization.
2：0, folded and purified, and attached to supported lipid bilayers (SLBs) via cholesterol-modified oligonucleotides. Diffusion is measured using single-particle tracking with a custom-built TIRF microscope, and super-resolution imaging is performed with DNA-PAINT microscopy. Salt concentrations (NaCl and MgCl2) are varied to control diffusion and immobilization. Sample Selection and Data Sources:
2. Sample Selection and Data Sources: DNA nanostructures include monomers and triskelia assembled from them, using scaffold P7650 and staple oligonucleotides. Lipid bilayers are formed from DOPC on glass or mica substrates.
3：List of Experimental Equipment and Materials:
Equipment includes a custom-built TIRF microscope, IX71 inverted microscope (Olympus), JEM-1011 transmission electron microscope (JEOL), and software like ImageJ with MosaicSuite plugin and Picasso for analysis. Materials include DNA oligonucleotides from Eurofins Genomics and Biomers, lipids from Avanti Polar Lipids, and buffers with varying ion concentrations.
4：Experimental Procedures and Operational Workflow:
SLBs are prepared by sonicating lipid solutions, incubating with cholesterol anchors, and adding DNA nanostructures. Diffusion is tracked under different salt conditions. For assembly, trimerization and multimerization oligonucleotides are added, followed by buffer exchanges for immobilization and DNA-PAINT imaging with imager strands.
5：Data Analysis Methods:
Diffusion coefficients are calculated from particle tracks using MosaicSuite. Super-resolution images are reconstructed with Picasso software, using Gaussian fitting and drift correction. Statistical analysis includes measuring nearest neighbor distances and counting monomers in clusters.
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IX71 inverted microscope
IX71
Olympus
Used for imaging DNA nanostructures under green illumination for diffusion measurements.
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JEM-1011 transmission electron microscope
JEM-1011
JEOL
Used for TEM imaging of DNA origami structures to confirm folding and assembly.
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TIRF microscope
custom-built
Used for single-particle tracking and super-resolution imaging of DNA nanostructures on lipid membranes.
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ImageJ software
Used with MosaicSuite plugin for analyzing particle tracks and diffusion coefficients.
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Picasso software
Used for super-resolution image reconstruction from DNA-PAINT data, including drift correction and Gaussian fitting.
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CaDNAno software
2.0
Used for designing DNA origami structures.
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sticky-Slide VI
0.4
ibidi
Used as a channel for preparing supported lipid bilayers and conducting experiments.
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Freeze 'N Squeeze DNA Gel Extraction Spin Columns
Biorad
Used for purifying DNA nanostructures from agarose gels.
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SYBR safe
Thermofisher
Used for pre-staining agarose gels for DNA visualization.
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DOPC lipid
18:1 (Δ9-Cis) PC
Avanti Polar Lipids
Used to form supported lipid bilayers for membrane experiments.
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Atto 647N dye
Eurofins Genomics
Used for fluorescent labeling of DNA nanostructures for tracking.
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Atto 655 dye
Used on imager strands for DNA-PAINT super-resolution imaging.
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cholesterol-TEG modified oligonucleotide
Biomers
Used to anchor DNA nanostructures to lipid membranes.
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scaffold DNA
P7650
Used as the backbone for folding DNA origami structures.
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