Programmable nucleic acid nanoparticles boost vaccine immune responses as effectively as standard adjuvants in mice while avoiding the autoimmune risks that limit existing alternatives.
(Nanowerk Spotlight) Every vaccine faces the same fundamental challenge: getting the immune system to pay attention. Antigens alone often fail to provoke responses strong enough to confer lasting protection. Adjuvants solve this problem by amplifying immune activation, but the options available to vaccine developers remain surprisingly limited. Aluminum salts have dominated since the 1920s.
Newer alternatives like Toll-like receptor agonists show potent activity but carry serious risks, including the potential to trigger autoimmune reactions that cause the body to attack its own tissues. This safety concern has restricted most such compounds to topical creams rather than injectable vaccines.
The rise of nucleic acid therapeutics has opened new possibilities. RNA and DNA molecules can now silence genes, edit genomes, and encode proteins for translation inside cells. Messenger RNA (mRNA) vaccines proved their worth during the COVID-19 pandemic.
Yet therapeutic nucleic acids remain difficult to work with. They degrade quickly in biological fluids, struggle to enter cells efficiently, and can trigger unwanted inflammation. The immune system treats foreign nucleic acids as danger signals, activating pattern recognition receptors that evolved to detect viral invaders.
What if this liability could become an asset? By folding nucleic acids into precise three-dimensional architectures, scientists can create structures that engage immune sensors in controlled ways. These nucleic acid nanoparticles assemble from multiple strands that interlock like pieces of molecular origami. Varying their size, shape, and chemical composition changes how the immune system responds.
The spectrum of modifications, their occurrence, relative nuclease resistance, thermal stabilities, and immunostimulation. In total, nine different compositions of NANPs were tested: DNA cubes (DNA), DNA cubes with backbone phosphorothioate modifications (PS), and DNA cubes containing ssUs in the corners (D-U). RNA cubes with hydroxyl group at 5’ end (5’OH), RNA cubes with triphosphate group on 5’ end (RNA). The rest of RNA cubes had 5’-triphosphates and chemical modifications at ribose, where 2’-hydroxyl was substituted with either fluorine (2’F) or 2’-O-methyl (2’OMe) groups or uridine is substituted with pseudouridine (ψ). (Image: Reproduced from DOI:10.1002/adfm.202515585, CC BY) (click on image to enlarge)
The work represents a highly collaborative interdisciplinary effort, bringing together researchers from the University of North Carolina at Charlotte, the Frederick National Laboratory for Cancer Research, the University of Virginia, Northeastern University, and other institutions. Their expertise spans chemistry, nanotechnology, immunology, computational biology, histopathology, and genomics.
“We constructed cube-shaped nanoparticles from six interlocking strands, keeping the three-dimensional architecture constant while varying chemical composition across nine variants,” Prof. Kirill A. Afonin at the University of North Carolina at Charlotte, tells Nanowerk. “These included standard DNA cubes, DNA with phosphorothioate backbone modifications that swap oxygen for sulfur to resist enzymatic degradation, and DNA containing uracil bases at the corners. RNA variants included unmodified cubes with triphosphate groups at the 5′ end, cubes with only hydroxyl groups at that position, and cubes modified with pseudouridine, 2′-fluoro groups, or 2′-O-methyl groups on the sugar ring.”
Physical testing revealed clear structure-property relationships. Modified RNA cubes exhibited melting temperatures between 55 °C and 62 °C, higher than unmodified versions. Cubes bearing 2′-fluoro, 2′-O-methyl, or pseudouridine modifications survived longer when exposed to degrading enzymes in serum.
Immune response profiling in human blood cells yielded the study’s central findings. When delivered into cells at 10 nM concentrations, unmodified RNA cubes carrying 5′ triphosphate groups triggered robust production of type I interferons after 24 hours. These signaling molecules, which include interferon-alpha, interferon-beta, and interferon-omega, coordinate antiviral defenses throughout the body. The cubes also induced type III interferon-lambda. DNA cubes activated the same pathways but less potently.
Afonin points out that chemical modifications dramatically altered this picture: “Cubes made with 2′-O-methyl RNA or phosphorothioate DNA triggered essentially no interferon production. Those bearing 2′-fluoro or pseudouridine modifications showed significantly reduced activity. The choice of lipid carrier also mattered: a formulation called DOTAP/DOPE produced weaker interferon responses than Lipofectamine across all cube types, providing another dial for adjusting immune activation.”
Hybrid cubes containing mixtures of modified and unmodified strands offered even finer control. Adding just one 2′-fluoro strand to an otherwise unmodified RNA cube cut its immunostimulatory potential by more than half. Different strand ratios produced intermediate effects, allowing researchers to dial in specific activity levels by blending chemistries.
Single-cell RNA sequencing revealed that cube-treated immune cells behaved like those responding to viral infection. The nanoparticles expanded populations of T cells, B cells, and dendritic cells while reducing monocyte numbers. Gene expression analysis identified 1,565 differentially expressed genes, with interferon signaling and antiviral response pathways prominently affected.
Mechanistic studies pinpointed which immune sensors recognize the cubes. Reporter cell lines showed that unmodified RNA cubes strongly activated RIG-I, a cytosolic receptor that detects 5′ triphosphate groups characteristic of certain viral genomes. Modified cubes activated RIG-I far less effectively. Among endosomal Toll-like receptors, only TLR7 responded substantially to unmodified RNA cubes. Computational modeling using AlphaFold3 structure prediction and molecular dynamics simulations supported these findings, revealing that 5′ triphosphate groups enhance binding to TLR7 while certain sugar modifications weaken interactions with RIG-I.
“The critical test came in mice,” says Afonin. “Animals received the model antigen ovalbumin alone, with standard alum adjuvant, or with RNA cubes complexed with DOTAP/DOPE lipids. After 21 days, mice receiving RNA cubes produced antibody levels equivalent to those receiving alum, the clinical gold standard for vaccine adjuvants.”
Safety testing addressed the concern that has blocked other nucleic acid immunostimulants from systemic use. The team administered cubes to mice genetically predisposed to autoimmune disease and monitored them for 20 weeks. Cubes alone caused no increased mortality, no kidney damage, and no elevation of antibodies targeting the body’s own DNA.
Even combined with pristane, a chemical that induces lupus-like disease in this mouse strain, the cubes did not worsen outcomes. Traditional Toll-like receptor agonists, by contrast, carry autoimmune risks that have confined them to topical applications.
Further safety assessment employed organ-on-chip technology, growing three-dimensional intestinal epithelial tubes on microfluidic devices to model barrier function. Cube delivery caused only temporary decreases in barrier integrity that recovered within 72 hours, suggesting compatibility with mucosal vaccine administration routes.
“Our findings position nucleic acid nanoparticles as a programmable immunotherapy platform with a key advantage: the ability to match clinical adjuvant potency without triggering autoimmunity,” Afonin concludes. “Selecting from a menu of chemical modifications will allow researchers to tune thermal stability, enzymatic resistance, and immune activation independently. Mixing modified and unmodified strands provides additional precision.”
This combination of modularity and safety opens a path toward next-generation vaccines for infectious diseases and cancer, built from the same molecular alphabet that encodes life itself.
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Kirill Afonin (University of North Carolina at Charlotte)
, 0000-0002-6917-3183 corresponding author
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