Mandal, S.; Chandrasekaran, A. R.; Maiti, P. K.

Designing biostable DNA nanocarriers for precise therapeutic delivery remains a key challenge in DNA nanotechnology due to susceptibility to nuclease degradation. Multi-stranded DNA nanostructures, such as Paranemic crossover (PX) DNA, show enhanced biostability compared to native duplex DNA due to their unique topology. However, the molecular origin of their exceptional nuclease resistance is still unknown. Using atomistic MD simulation and enhanced sampling, we uncover the molecular origins of PX-DNA\'s superior resistance over JX-DNA and dsDNA. Our findings reveal that PX-DNA\'s six crossover points induce an overtwisted helix and narrower minor groove, leading to reduced DNase I binding affinity ( ~ +5 kcal/mol for PX-DNA vs. ~ -17 kcal/mol for dsDNA). Mechanical properties, such as the stretch modulus ({gamma}G), confirm enhanced structural rigidity of PX-DNA (4805 pN), while residence time calculations using {tau}-RAMD further highlight shorter residence time of DNase I nuclease on PX-DNA. This is the first theoretical study to explore crossover-dependent biostability, mechanical properties, and residence time of PX/JX nanostructures in the presence of a nuclease using enhanced sampling. Our findings highlight that strategically positioned crossover points can regulate DNA stability against nuclease degradation at the nanoscale and lead to better design of biostable DNA nanostructures for medicinal applications.