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

Abstract

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 50 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.

PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy

“…16 The strength of the electrostatic attraction can be modified by the presence of additional ions and chelating agents within the imaging buffer. 17,18 Other methods to facilitate DNA absorption include the modification of surface chemistry using silanes, 15 the formation of partially positively charged lipid bilayers 9,19 and the electrostatic adsorption of positively charged polymers such as poly-L-lysine (PLL). 20,21 The aforementioned approaches are often adopted for the study of DNA-protein binding using AFM.…”

A DNA-based optical nanosensor forin vivoimaging of acetylcholine in the peripheral nervous system

“…The injection of ACh was repeated 5 times. AFM measurement was conducted following literature precedents 56 . All buffers were freshly prepared and filtered through 0.22 µm filters on the experiment day.…”

AutoSmarTrace: Automated Chain Tracing and Flexibility Analysis of Biological Filaments

“…Additionally, we also traced a sample of AFM images of DNA with the automated and manual methods. These results were both compared to the value of persistence length independently obtained through a separate method of manual tracing (5).…”

“…At present, there is no standard technique for determining backbone coordinates from AFM images. Manual chain tracing is frequently done when image sets are not too extensive (5), but for larger numbers of images, manual tracing can become cumbersome. Furthermore, it is possible that there is variation in backbone coordinates selected by different users, which could lead to biased results.…”