Weakly Ionized Interface Breaks the Poly(A) Terminal Length Constraint in Freezing Directed Spherical Nucleic Acids

Abstract Non-thiolated spherical nucleic acids (SNAs) are widely used in biochemical applications, yet their synthesis remains constrained by sequence-dependent DNA adsorption onto gold nanoparticles (AuNPs). In freezing method, efficient adsorption typically requires a long poly(A) (≥10 nt), although the origin of this requirement remains unclear. Here, we show that this limitation arises primarily from the highly charged interface of citrate-stabilized AuNPs rather than the freezing process itself. Replacing citrate with weakly ionized ascorbic acid reduces interfacial electrostatic repulsion and promotes DNA adsorption, enabling stable SNAs with terminal fragments containing only two adenines (≥2 nt) and breaking the Poly(A) length limitation in freezing directed spherical nucleic acids. In this system, adenine accessibility, rather than sequence position or distal composition, governs adsorption behavior. Benefiting from the weakly ionized interfacial interactions, the resulting SNAs exhibit a high DNA packing density, imparting excellent colloidal stability (stable for 50 days in 0.1 M NaCl) and reproducible plasmonic properties (RSD = 5.1%). These findings establish interfacial interaction engineering as a general strategy for sequence-independent construction of non-thiolated SNAs.