Chirality of DNA supercoiling assigned by scanning force microscopy

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• Plasmid DNA, such as pBR322, is crucial in molecular biology and genetic engineering.
• Understanding the structural conformation of circular DNA is essential for various biological processes.
• Previous studies utilized electron microscopy to visualize DNA structures, but high-resolution imaging in solution remains challenging.

Purpose of the Study:

• To obtain reproducible scanning force microscopy (SFM) images of pBR322 plasmid molecules in a 1-propanol environment.
• To characterize the structural states and supercoiling patterns of pBR322 plasmids.
• To develop a method for assigning the chirality of local supercoiling in individual DNA molecules.

Main Methods:

• Utilized scanning force microscopy (SFM) to image pBR322 plasmid molecules.
• Performed imaging in a 1-propanol solution to maintain DNA structure.
• Applied stratigraphic analysis to SFM height data for detailed structural characterization.

Main Results:

• Reproducible SFM images of pBR322 plasmid DNA were successfully obtained.
• The majority of observed plasmid molecules were in a coiled state.
• Supercoiled molecules exhibited branched or unbranched interwound superhelix structures, consistent with electron microscopy data.
• Stratigraphic analysis allowed for the assignment of local supercoiling chirality for individual molecules.

Conclusions:

• SFM is a viable technique for imaging plasmid DNA structure in solution.
• pBR322 plasmids predominantly exist in coiled conformations with complex superhelical structures.
• Stratigraphic analysis of SFM data provides a novel approach to determine DNA supercoiling chirality.