Rational Design of DNA Nanostructures as TLR9 Agonists

TLR9 agonists hold significant potential in vaccine development and cancer immunotherapy. While synthetic CpG oligodeoxynucleotides (ODNs) have been employed in immunotherapies, their clinical application remains constrained by dose-limiting toxicity and metabolic instability. DNA nanotechnology provides a promising strategy for the precise modulation of ligand-TLR9 interactions. In this study, DNA nanostructure-based TLR9 agonists were rationally designed by optimizing the spatial orientation of 5′-TCG ligands based on the two DNA-binding sites revealed in the TLR9 receptor crystal structure. Fine-tuning DNA nanostructure configurations enabled controllable modulation of TLR9 activation with structural flexibility identified as a critical determinant. Notably, the oligomerization of DNA nanostructures markedly enhanced TLR9 stimulation efficacy, establishing a paradigm for the rational design of TLR9 agonists. These findings advance innovative approaches for developing next-generation immunotherapeutic agents.