Thermal stability and dynamics of ssDNA loaded adeno-associated viral capsids: A coarse-grained molecular dynamics simulations

The study investigated the structural breakdown as a function of temperature and potential genome release of adeno-associated virus (AAV) in an aqueous environment through coarse-grained molecular dynamics (CG-MD) simulations. The factors and mechanisms influencing capsid stability using an AAV8 capsid loaded with single-stranded DNA (ssDNA) were studied across a range of 300 K, 338 K and 363 K temperatures. The simulations, which spanned over 10 microseconds (µs), successfully captured the temperature-dependent structural changes in the full-capsid. At higher temperatures, ssDNA-filled capsids exhibited weakening of their compact structure, resulting in increased instability. This destabilization was reflected in larger conformational shifts among protein subunits and greater fluctuations in amino acid residues, both contributing to capsid disruption. The results showed that elevated temperatures significantly compromise the stability of temperature-sensitive protein subunits and promote the thermal expansion of ssDNA, ultimately leading to the collapse of the 5-fold pentamer structure. Moreover, the asymmetry of ssDNA may promote distortions from a spherical capsid shape, further contributing to structural failure. The accuracy of the CG-MD methodology was validated by comparison with previous experimental and simulation data, including full capsid diameters and internal pressure measurements. Overall, this computational framework, supported by validation efforts, advances our understanding of AAV structural stability, capsid breakage mechanisms, and the potential for ssDNA leakage.