Comparative studies of DNA-based monoclonal antibody mimics

Monoclonal antibodies (mAbs) are a leading class of biological drugs known for their high target specificity and low dosage requirements. However, their production is costly and laborintensive, they require strict storage conditions due to degradation risks, and they often elicit immune responses that can reduce efficacy. In contrast, nucleic acid nanoparticles (NANPs) present a promising alternative, offering broader target accessibility, enhanced stability, prolonged shelf-life at ambient conditions, cost-effective, and rapid assembly. In this study, we designed and assembled a library of 18 NANPs functionalized with prostate-specific membrane antigen (PSMA)-binding aptamers to mimic the structural and functional properties of mAbs including monomeric, dimeric, and pentameric isotypes. We further characterized the physicochemical properties of DNA polygons by the means of electrophoretic mobility shift assay (EMSA), dynamic light scattering (DLS), and resistance to enzymatic degradation. EMSA and DLS experiments confirmed high assembly efficiency across all isotype mimics and resulted in a homogeneous solution. The polygons remained stable in 10% FBS, permitting further in vitro experiments with various mammalian cell lines. The trivalent NANP-mAb exhibited high assembly efficiency (>90%), compact size, and low polydispersity, making it the optimal candidate for binding studies, while aptamer functionalization modestly increased diameter and surface charge without compromising stability. Binding assays preliminarily confirmed that the constructs retained specific recognition of PSMA, with the trivalent A12-decorated NANP-mAb showing micromolar affinity (KD = 9.99 × 10⁻⁶ M) and six-fold selectivity over a control protein. Comparative studies of multiple aptamers highlighted that A12 and its truncated variant had the strongest binding within the NANP format, emphasizing that aptamer performance is context- and presentation-dependent. The DNA-based NANP platform offers precise programmability, modularity, chemical stability, and controllable valency, enabling targeted designs and combinatorial strategies beyond the capabilities of conventional antibodies.