Designing biostable DNA nanocarriers for targeted therapeutic delivery remains a key challenge in DNA nanotechnology due to their susceptibility to nuclease degradation. Multi-stranded DNA nanostructures, such as paranemic crossover (PX-DNA), exhibit significantly enhanced biostability compared to native dsDNA, owing to their unique crossover-dependent topological features. However, a complete molecular understanding of the mechanism behind this exceptional nuclease resistance of PX-DNA nanostructures is still lacking. In this study, we use atomistic molecular dynamics simulations to investigate the interaction behaviour of DNase I nuclease and uncover the molecular origin of the enhanced resistance exhibited by crossover-rich PX-DNA compared to native dsDNA. Our simulation results reveal that the six crossover points in PX-DNA induce an over-twisted (∼35°) helix and narrower minor grooves, reducing DNase I binding affinity (i.e., ∼+5 kcal mol-1 for PX-DNA vs. ∼-17 kcal mol-1 for dsDNA). The stretch modulus (γG) calculations further confirm enhanced mechanical stiffness of PX-DNA (∼4804 pN) compared to that of dsDNA (∼1845 pN). These findings highlight how strategically positioned crossover sites can significantly modulate DNA stability against nuclease degradation at the nanoscale, offering a molecular framework for designing robust, biostable DNA nanostructures for targeted therapeutic delivery applications.
Mandal et al. (Thu,) studied this question.