Easier AFM imaging of DNA
Gentle processing conditions yield DNA image
US-based researchers from JILA - a joint institute of the University of Colorado Boulder and NIST - have developed a fast and simple sample preparation method that enhances atomic force microscopy of DNA.
The five-minute process extends the DNA’s configuration so that eight times more of the molecule can be analysed as compared to previous methods.
“We expect this new sample preparation method to pave the way to tuning the binding strength of DNA to a surface, which should facilitate studying the dynamics of protein-DNA complexes,” says NIST/JILA Fellow Tom Perkins.
JILA’s process includes pre-soaking a mica binding surface in a nickel-salt solution, then gently rinsing and drying it.
The researchers then binding the DNA to the mica in a solution containing magnesium chloride and potassium chloride.
As in a cell, these salt conditions preserve the properties of proteins binding to DNA.
After the protein-DNA complexes bind to the mica, the substrate and sample are then rinsed with a solution containing nickel-chloride, which traps the DNA structure by increasing the DNA-mica interaction strength.
As Perkins writes in ACS Nano: “The atomic flatness of mica makes it the preferred substrate for high signal-to-noise ratio [AFM] imaging, but the negative charge of mica and DNA hinders deposition.”
“But we have have developed this process to bind DNA to mica in a buffer containing both magnesium chloride and potassium chloride that resulted in high signal-to-noise ratio images of equilibrated DNA in liquid,” he says.
Following sample preparation, Perkins and colleagues were able to produce high-quality images over a wide range of salt concentrations, including concentrations akin to those found in a cell.
This was previously thought impossible because different salts ordinarily compete to attach the DNA to the surface or interfere with that attachment.
High-resolution images revealed the DNA’s iconic double helix structure.
JILA’s new sample preparation process improves imaging of DNA in liquid, as shown in this colourised, 3D view of the molecule. The gentle processing conditions yield an image of DNA at its correct length and height (680 and 2 nm respectively). The iconic double helix shape of DNA can be seen (inset). Both images were filtered to remove extraneous features. (Image: Heenan/JILA)
As the researchers highlight, for the first time in liquid, their method produced AFM images of DNA bound to the flat surface without any alterations to mechanical properties such as width, length and native backbone stiffness.
The enhanced images of DNA-protein complexes will help researchers understand new details of processes such as DNA repair and cellular metabolism.
Research is published in ACS Nano.