We use atomic force microscopy (AFM) nano-indentation method to measure DNA mobility in the capsid. The mobility of DNA packaged inside the HSV-1 capsid is measured by recording the force resisting the capsid indentation when the AFM tip is brought into contact with the DNA-filled capsid in solution. Force resisting the AFM tip indentation of the capsid is recorded as the force-distance curve. Capsid deformation leads to displacement of water molecules hydrating the DNA and interhelical sliding, occurring with compression of the packaged DNA strands. This constitutes our approach for analyzing intracapsid DNA mobility at a single-particle level. Using AFM combined with ITC, we have recently shown that restricted intra-capsid DNA mobility can be characterized by a solid-like mechanical response to nano-indentation of a DNA-filled capsid. At the same time, DNA packaged in the capsid can undergo a mechano-structural transition from a solid- to a fluid-like state. This transition is induced by increasing the temperature (which affects DNA bending stress and packing defects) or by varying external ionic conditions (which affects DNA-DNA repulsive interactions and overall genome stress in the capsid). In the fluid-like state, the interstrand DNA-DNA friction of the encapsidated genome is significantly reduced due to an increase in distance between packaged DNA strands occurring with local DNA disordering. These AFM observations suggest that dynamics of DNA ejection can be affected by a transition in mobility of the encapsidated genome. We continue investigate the effects of this mechano-structural DNA transition on DNA ejection dynamics from viruses and viral replication.

Figure: AFM nano-indentation analysis of encapsidated DNA mobility. (a) AFM image of HSV-1 capsid in buffer. The inset shows a cross-section profile along the dashed line. Scale bar, 150 nm. (b) AFM image of HSV-1 capsid after breaking. Scale bar, 150 nm. (c) Zoomed-in image of HSV-1 C-capsid with individual hexons observed on the capsid surface. Scale bar, 30 nm. (d) Force-distance curves for glass substrate and for HSV-1 C-capsid. An abrupt drop in the force-distance curve signifies breaking of the capsid.