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Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging
Nicholas A W Bell1, Justin E Molloy1
1The Francis Crick Institute, London NW1 1AT, UK.
Nucleic Acids Research
|April 30, 2022
Summary
Researchers developed a fast DNA assembly method using golden gate rules for single-molecule biophysics. This technique efficiently creates diverse DNA constructs, improving DNA-protein interaction studies.
Area of Science:
- Biophysics
- Molecular Biology
- Biochemistry
Background:
- Single-molecule techniques like optical tweezers and fluorescence imaging are crucial for studying DNA and DNA-protein interactions.
- Efficient methods for creating custom DNA structures with specific labels are needed for these biophysical studies.
Purpose of the Study:
- To develop a simple, fast, and adaptable technique for fabricating diverse DNA constructs for single-molecule biophysics.
- To demonstrate the utility of this method for creating torsionally-constrained DNA and DNA hairpin structures.
Main Methods:
- Utilized golden gate assembly rules to combine PCR amplicons and synthetic oligonucleotides.
- Fabricated torsionally-constrained duplex DNA up to 10 kbp and various DNA hairpin structures.
- Investigated tethering to a cross-linked antibody substrate to enhance measurement lifetime under high force.
Main Results:
- Achieved high-yield fabrication of diverse DNA constructs.
- Successfully created long (up to 10 kbp) torsionally-constrained DNA duplexes.
- Demonstrated enhanced measurement stability using antibody-based surface tethering.
Conclusions:
- The developed golden gate assembly method is a rapid and versatile tool for creating DNA constructs for single-molecule biophysics.
- This technique streamlines the preparation of DNA substrates, facilitating advanced studies of DNA and DNA-protein interactions.
- Enhanced tethering strategies improve the reliability and duration of single-molecule measurements under demanding conditions.
