Imaging DNA Equilibrated onto Mica in Liquid Using Biochemically Relevant Deposition Conditions

摘要

For over 25 years, imaging of DNA by atomic force microscopy has been intensely pursued. Ideally, such images are then used to probe the physical properties of DNA and characterize protein-DNA interactions. The atomic flatness of mica makes it the preferred substrate for high signal-to-noise ratio (SNR) imaging, but the negative charge of mica and DNA hinders deposition. Traditional methods for imaging DNA and protein-DNA complexes in liquid have drawbacks: DNA conformations with an anomalous persistence length (p), low SNR, and/or ionic deposition conditions detrimental to preserving protein-DNA interactions. Here, we developed a process to bind DNA to mica in a buffer containing both MgCl2 and KCl that resulted in high SNR images of equilibrated DNA in liquid. Achieving an equilibrated 2D configuration (i.e., p 50 nm) not only implied a minimally perturbative binding process but also improved data quality and quantity because the DNA's configuration was more extended. In comparison to a purely NiCl2-based protocol, we showed that an 8-fold larger fraction (90%) of 680-nm-long DNA molecules could be quantified. High-resolution images of select equilibrated molecules revealed the right-handed structure of DNA with a helical pitch of 3.5 nm. Deposition and imaging of DNA was achieved over a wide range of monovalent and divalent ionic conditions, including a buffer containing SO mM KCl and 3 mM MgCl2. Finally, we imaged two protein-DNA complexes using this protocol: a restriction enzyme bound to DNA and a small three-nucleosome array. We expect such deposition of protein-DNA complexes at biochemically relevant ionic conditions will facilitate biophysical insights derived from imaging diverse protein-DNA complexes.