Atomistic insights into spermidine-mediated stabilization of paranemic crossover DNA motifs

The polyamine spermidine (Spd³⁺), which is naturally abundant in cells, influences the architecture and stability of nucleic acid assemblies through multivalent electrostatic interactions. In this study, we investigated how Spd³⁺ affects the paranemic crossover (PX) motif, a key structural element in DNA nanotechnology. Quantum mechanical calculations (HF/6‑31G*) combined with RESP‑derived partial charges were used to optimize the molecular geometry of Spd³⁺ and obtain a reliable electrostatic description. We then performed all-atom molecular dynamics (MD) simulations in GROMACS under two ionic conditions, neutralizing the systems with either Na⁺ or Spd³⁺. Radial distribution functions (RDFs) and minimum‑distance analyses showed that Spd³⁺ binds more strongly and persistently to the DNA backbone than Na⁺. Energy decomposition revealed more favorable electrostatic and van der Waals interactions in the presence of Spd³⁺, whereas Na⁺ exhibited weaker binding. Mean‑square displacement (MSD) analysis further confirmed the localized behavior of Spd³⁺ compared with the higher mobility of Na⁺. Global structural metrics, including principal component analysis (PCA) and root‑mean‑square deviation (RMSD), indicated a more confined conformational landscape under Spd³⁺ conditions. Analysis of base‑pair step parameters characterized the local helical geometry within the crossover region. In parallel, backbone fluctuation analysis showed reduced flexibility in the presence of Spd³⁺. Overall, these computational findings suggest that spermidine enhances the structural stability of the PX motif and provide molecular-level insights into the potential robustness of DNA‑based nanomaterials.