Reprogrammable RNA nanoparticles activate only in cancer cells to deliver targeted gene-silencing therapy while avoiding off-target effects, immune activation, and unnecessary drug exposure.
(Nanowerk Spotlight) Cancer therapies based on RNA interference (RNAi) promise highly targeted gene silencing, but that precision often comes at a cost. Once inside the body, these molecules must reach the right tissue, enter the right cells, and hit the right gene—all without triggering the immune system or harming healthy cells. Most RNAi drugs rely on constant exposure to maintain efficacy, and even then, off-target effects and delivery complications remain major hurdles.
Current efforts to improve targeting often involve modifying the RNA payload or attaching ligands to nanoparticles. But these strategies are static—they don’t change in response to local cellular conditions. What if the therapeutic response could be conditional, triggered only when a diseased cell signals its presence?
That’s the question a research team, led by the Afonin Lab at UNC Charlotte, have tackled with a new class of molecular devices called reconfigurable nucleic acid nanoparticles, or recNANPs.
Each recNANP is constructed as a modular unit composed of both sensing and therapeutic elements, enabling it to respond to specific genetic signals in cancer cells. As Prof. Afonin explains to Nanowerk:
“Each recNANP is assembled from four short strands of DNA and RNA. The structure integrates a diagnostic module, which binds to a target mRNA sequence, and a therapeutic module composed of split Dicer-substrate RNAs. These RNA fragments only become functional after the nanoparticle undergoes a conformational change triggered by contact with a cancer-specific RNA, in this case, the KRAS G12D mutation. When activated, the strands reassemble to form a duplex that can be processed by the cell’s own machinery into active siRNAs, which then suppress the expression of pro-survival genes such as Survivin and BCL-2.”
This modular architecture allows the particles to remain inert in healthy cells and only activate in the presence of disease-specific mRNA. Once triggered, the response is localized, durable, and designed to selectively knock down key genes that support tumor survival.
Modular four-stranded reconfigurable nucleic acid nanoparticles (recNANPs) for conditional activation of therapeutic RNAi responses upon intracellular interaction with a target mRNA. (A) Diagnostic – working principle of molecular beacons (MBs). (B) Treatment – working principles of split RNAi inducers. (C) Logic gate rules for input and output products wherein recNANPs act as a simple NOT logic gate. In (D), 2D schematics of recNANPs activation and (E) predicted 3D structures are shown for all constructs. (Image: reprinted from DOI:10.1002/adfm.202508122, CC BY) (click on image to enlarge)
Unlike conventional delivery systems, the recNANPs function according to a simple logic rule: they only release their therapeutic cargo if the disease marker is present. This logic—akin to a NOT gate in computing—ensures that healthy cells are ignored.
However, the system’s precision has limits. The stem-loop structure used to detect KRAS G12D does not sharply distinguish between mutant and wild-type sequences. Instead, activation depends primarily on how strongly KRAS is expressed. This makes the platform best suited to disease contexts where target genes are overexpressed, rather than merely mutated.
To test this behavior, the researchers introduced recNANPs into two cell lines: PANC-1, which harbors the KRAS G12D mutation, and HEK-293FT, which expresses wild-type KRAS at lower levels. In PANC-1 cells, the particles successfully reconfigured and released therapeutic siRNAs, resulting in strong downregulation of target genes. In contrast, the nanoparticles remained largely inactive in the wild-type cells. The same pattern held in three-dimensional spheroid models, where the particles showed deep penetration and specific activity in tumor-like structures.
In addition to demonstrating target selectivity, the study compared recNANPs to traditional Dicer-substrate RNAs. While the latter entered cells more efficiently, they also showed greater toxicity and a shorter duration of effect. By contrast, recNANPs provided more sustained silencing with lower immune activation. The team also tested the particles in combination with chemotherapeutic agents such as doxorubicin and cisplatin.
Even at reduced drug concentrations, co-treatment with recNANPs significantly lowered cell viability, suggesting that the particles could amplify the effects of existing cancer therapies while reducing their side effects.
Immune compatibility remains a central challenge for all nucleic acid-based treatments. To assess this, the researchers exposed human peripheral blood mononuclear cells to recNANPs delivered using two types of lipid carriers: Lipofectamine and DOTAP. The DOTAP formulation induced minimal cytokine release and avoided triggering major pattern recognition receptors such as TLR3, TLR7, TLR9, and RIG-I.
Lipofectamine, on the other hand, produced a modest interferon response, highlighting how delivery vehicle design influences immune sensing. Mechanistic studies using reporter cell lines confirmed that the particles themselves were largely invisible to innate immune surveillance, likely due to their DNA/RNA hybrid composition and compact structure.
To demonstrate the platform’s flexibility, the team reprogrammed the therapeutic module to target a second gene, BCL-2, and achieved comparable results. Swapping the recognition loop and therapeutic RNA strands did not require redesigning the core scaffold, underscoring the modularity of the system. This plug-and-play design could, in principle, be adapted to a wide range of gene targets associated with cancer, viral infection, or inflammatory disease.
The group’s earlier work (“Advanced SAXS-MD framework reveals RNA nanoparticle dynamics in solution”) combining small-angle X-ray scattering with molecular dynamics simulations to study RNA nanoparticle conformations in solution helped inform these design strategies by revealing how strand architecture and dynamic flexibility influence structural behavior.
“While challenges remain – particularly in improving discrimination between similar sequences and ensuring stable delivery under physiological conditions – the recNANP strategy introduces a new level of conditional logic into RNA therapeutics,” Afonin concludes. “By embedding both recognition and response into a single structure, the particles act less like static drugs and more like autonomous molecular devices, programmed to act only in the presence of disease.”
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