Long DNA constructs to study helicases and nucleic acid translocases using optical tweezers

Area of Science:

• Molecular Biology
• Biophysics
• Biochemistry

Background:

• Single-molecule biophysics requires homogeneous long DNA with controlled sequences for studying DNA-protein interactions.
• Lambda phage DNA is traditionally used but lacks customization options.

Purpose of the Study:

• To provide a simple protocol for generating custom DNA constructs (~25kbp) for single-molecule experiments.
• To enable the production of specific DNA structures like single-stranded DNA (ssDNA), ssDNA-dsDNA hybrids, and DNA constructs with flaps.
• To facilitate surface immobilization of DNA constructs for advanced imaging techniques.

Main Methods:

• Cloning of large plasmids (~25kbp) with bespoke sequence content.
• Generation of long single-stranded DNA (ssDNA) molecules, ssDNA-dsDNA hybrids, and DNA constructs with flaps.
• Modification of DNA construct ends for surface binding and immobilization.
• Application of dual optical tweezers and confocal microscopy for imaging.

Main Results:

• A detailed and accessible protocol for producing custom DNA constructs is established.
• The protocol allows for the creation of versatile DNA substrates relevant for studying DNA helicases and translocases.
• Demonstrated successful application in studying human DNA helicase B (HELB) activity.

Conclusions:

• The described techniques are simple, versatile, and adaptable for various single-molecule studies.
• This method provides researchers with custom DNA constructs for detailed investigation of DNA-protein interactions.
• The protocol is accessible to laboratories utilizing standard molecular biology techniques.