MultidimensionalQuantitative Analysis of BiologicalResponses to DNA Nanoassemblies with Defined Structure and SurfaceChemistry

Abstract DNA nanoassemblies (DNs) have emerged as programmable platforms for biomedical applications; however, a quantitative understanding of how structure and surface chemistry jointly govern their biological behavior remains limited. Here, we establish a multidimensional analytical framework to systematically quantify structure-surface-function relationships in DNs. Representative DNs with distinct structural architectures, including a one-dimensional rod-like six-helix bundle (6HB), a two-dimensional planar three-point star (3PS), and a three-dimensional tetrahedron (TDN), along with their cholesterol- and PEG20k-modified counterparts, were constructed and comparatively analyzed across serum stability, cellular uptake, inflammatory responses, and in vivo performance. Both structure and surface modification significantly influenced DN behavior. Cholesterol modification generally enhanced cellular uptake across BMDM, HEK293T, and HeLa cells to 1.09 to 3.34 times that of the corresponding unmodified DNs, while reducing serum stability by 44–70% after 24 h of incubation in 20% FBS. In contrast, PEG20k modification increased serum stability by 27–85% and attenuated inflammatory responses. Notably, identical surface modifications produced distinct biological effects across DNs of different structures, supporting a coupled rather than additive interplay between structure and surface chemistry. This work provides a quantitative basis for correlating DN design parameters with biological outcomes and establishes a generalizable analytical framework for matching DN structure and surface chemistry to application-specific biological requirements.