Self-Assembled Multivalent DNA Nanostructures for Noninvasive Intracellular Delivery of Immunostimulatory CpG Oligonucleotides

TLDR

DNA nanostructures self‑assemble from designed oligonucleotides, providing precise, uniform platforms for biosensing, imaging, and drug delivery. The study develops multivalent DNA tetrahedra functionalized with unmethylated CpG motifs. The CpG motifs on the nanostructures activate TLR9 in cells, triggering cytokine secretion. The compact, mechanically stable, noncytotoxic nanostructures resist nuclease degradation, enter macrophage‑like cells without transfection agents, and multivalent CpG motifs markedly amplify TLR9‑mediated cytokine secretion, indicating high efficacy for targeted drug delivery.

Abstract

Designed oligonucleotides can self-assemble into DNA nanostructures with well-defined structures and uniform sizes, which provide unprecedented opportunities for biosensing, molecular imaging, and drug delivery. In this work, we have developed functional, multivalent DNA nanostructures by appending unmethylated CpG motifs to three-dimensional DNA tetrahedra. These small-sized functional nanostructures are compact, mechanically stable, and noncytotoxic. We have demonstrated that DNA nanostructures are resistant to nuclease degradation and remain substantially intact in fetal bovine serum and in cells for at least several hours. Significantly, these functional nanostructures can noninvasively and efficiently enter macrophage-like RAW264.7 cells without the aid of transfection agents. After they are uptaken by cells, CpG motifs are recognized by the Toll-like receptor 9 (TLR9) that activates downstream pathways to induce immunostimulatory effects, producing high-level secretion of various pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12. We also show that multivalent CpG motifs greatly enhance the immunostimulatory effect of the nanostructures. Given the high efficacy of these functional nanostructures and their noncytotoxic nature, we expect that DNA nanostructures will become a promising tool for targeted drug delivery.