Atomic force microscopy imaging of double stranded DNA and RNA

Area of Science:

• Biophysics
• Materials Science
• Molecular Biology

Background:

• Atomic Force Microscopy (AFM) offers high-resolution surface imaging capabilities.
• Imaging long nucleic acid molecules like DNA and dsRNA presents challenges in sample preparation and stable surface attachment.
• Accurate measurement of nucleic acid length and structure is crucial for various biological and nanotechnological applications.

Purpose of the Study:

• To develop and describe a reliable procedure for imaging long DNA and double-stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM).
• To achieve stable binding of nucleic acid molecules to a mica substrate for consistent imaging.
• To validate the accuracy of length measurements obtained through the developed AFM procedure.

Main Methods:

• Chemically modifying freshly cleaved mica surfaces with 3-aminopropyltriethoxy silane under mild conditions.
• Utilizing Atomic Force Microscopy (AFM) for imaging prepared nucleic acid samples (intact lambda DNA, Hind III restriction fragments of lambda DNA, reovirus dsRNA).
• Performing imaging in both air and under water to assess sample stability and attachment strength.

Main Results:

• Striking AFM images of intact lambda DNA, DNA fragments, and dsRNA were obtained.
• The developed procedure resulted in stable nucleic acid attachment to the mica surface, allowing for repeated scanning.
• Measured contour lengths of DNA molecules were accurate to within a few percent.
• DNA image widths ranged from 20 to 80nm when imaged in air with commercial probes.

Conclusions:

• The described procedure enables routine and accurate length measurements of long DNA and dsRNA molecules using AFM.
• AFM is demonstrated as a valuable tool for simple measurements like length distribution of nucleic acids.
• Further improvements in substrate and sample preparation could lead to even higher resolution imaging.