Mechanistic Insights into the Stability of DNA Nanostructures: Effects of Structural Positioning, Chemical Modifications, and Crosslinking Strategies

State Key Laboratory of Digital Medical Engineering, School of Biological Sciences & Medical Engineering, Southeast University, Nanjing 210096, China

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Abstract

DNA nanotechnology enables the programmable construction of nanoscale architectures with high precision and has enabled applications in drug delivery, biosensing, and nanofabrication. However, the limited stability of DNA nanostructures under physiological conditions, particularly due to nuclease- mediated degradation and ionic fluctuations, remains a major challenge. In this work, we combine literature analysis with experimental validation to investigate the mechanisms governing the stability of DNA nanostructures. We focus on structural positioning, chemical modifications, and crosslinking strategies. Representative systems, including DNA double-crossover (DX), paranemic crossover DNA (PX), and DNA origami, are discussed to highlight structural influences on stability. Experimentally, we constructed DX and PX motifs and square DNA origami structures to evaluate stability under enzymatic conditions. Using exonuclease I/III and DNase I/FBS assays, we quantitatively compared the effects of chemical modifications and structural features on degradation behavior. We demonstrate that spatial heterogeneity plays a critical role, where exposed strand termini are more susceptible to enzymatic degradation, while internal regions are relatively protected. Furthermore, chemical modifications such as terminal modifications, 2′-O-methyl substitutions, and phosphorothioate backbone modifications significantly enhance nuclease resistance, with their effectiveness strongly dependent on positional distribution. Crosslinking strategies further improve structural rigidity and environmental tolerance. These findings provide a mechanistic framework for the rational design of stable DNA nanostructures and support their development for advanced biomedical applications.