Immunotherapies require novel type of delivery systems with specifically tailored adjuvants to activate immune responses. Among immune stimulators of microbial origin, oligodeoxynucleotides (ODNs) represent the most advanced potential adjuvants. ODNs are unmethylated single stranded DNA (ssDNA) sequences with CpG-motifs, which are able to activate the innate immune system by binding to TLR9 receptors. Adjuvant effects are optimized by maintaining ODNs and vaccine antigens in close proximity, which can be achieved by loading the immune stimulator and the antigen cargo to an appropriate carrier such as inorganic nanoparticles.<br/>Immobilisation of ODN immune stimulators onto the surface of nanoparticles while maintaining multivalent presentation to TLR9 receptors requires knowledge of their conformational properties. <i>De novo</i> modelling of ssDNA conformation, opposed to that of double stranded DNA, is challenging due to multiple reasons. ssDNA lacks stable structures and can only be described as an ensemble of interconverting conformations, thus methods for adequate sampling of the conformational space need to be applied. Force fields for simulation of DNAs were, however, developed by testing them mainly on duplex DNA and their ability to reproduce conformations of ssDNA is unclear. In this regard, modelling of ssDNA likely face similar challenges as modelling of Intrinsically Disordered Proteins.<br/>We set out to test the accuracy of existing DNA force fields using atomistic molecular dynamics (MD) simulations and NMR spectroscopy. Translational diffusion coefficients were obtained from diffusion experiments and compared to translational diffusion coefficients, radius of gyration and end-to-end distances calculated from MD simulations. Furthermore, we analysed secondary structure formation and descriptors of dynamic behaviour. Outcome of comparisons for different DNA force fields are discussed.

Investigations of immune stimulatory single stranded DNA by biomolecular simulations and NMR

ACS Fall 2021

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