Visualization of supercoiled DNA with atomic force microscopy in situ

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• Tertiary structure of supercoiled DNA is crucial for genetic functions.
• Environmental conditions, particularly ionic strength, can affect DNA supercoiling.
• Understanding DNA tertiary structure is key to comprehending genetic regulation.

Purpose of the Study:

• To investigate the impact of varying ionic conditions on the tertiary structure of supercoiled DNA.
• To visualize DNA supercoils in situ using high-resolution atomic force microscopy.
• To correlate DNA supercoil geometry with environmental ionic concentrations.

Main Methods:

• Applied atomic force microscopy (AFM) for high-resolution imaging of supercoiled DNA.
• Utilized an in situ sample preparation technique allowing imaging in buffer solutions without drying.
• Examined DNA morphology across a range of ionic strengths, from low to near-physiological concentrations.

Main Results:

• At low ionic strength, DNA molecules formed loosely interwound, irregularly shaped supercoils.
• In high-concentration, near-physiological salt solutions, plectonemic superhelices were observed.
• High ionic conditions led to superhelical loops separated by close helix-helix contacts.

Conclusions:

• The overall geometry of supercoiled DNA is dramatically dependent on ionic conditions.
• The formation of close contacts between DNA helices at physiological ionic strengths is a significant observation.
• These findings highlight the importance of ionic environment in modulating supercoiled DNA structure for biological functions.