How the University of North Carolina at Chapel Hill is enhancing RNAi potency with its new technology

Over the past decade, RNA interference (RNAi) technology has matured into a clinically validated therapeutic approach, with multiple small interfering RNA (siRNA) drugs delivering durable gene silencing in patients, particularly for liver-targeted indications. While these successes have ultimately validated RNAi as a clinically viable mechanism for precise gene silencing in humans, the structural constraints of conventional siRNAs continue to limit potency and restrict broader therapeutic reach.  

But now, a newly developed platform technology from The University of North Carolina at Chapel Hill (UNC-Chapel Hill) – a college that benefits from being located in North Carolina’s Research Triangle area – could potentially address these bottlenecks through precise DNA-based modifications at the 5′ end of the antisense strand, essentially offering a streamlined strategy to enhance RNAi potency and expand the scope and performance of RNAi therapeutics. 

The lowdown: What is RNAi technology?  

Before we dive into UNC-Chapel Hill’s new technology, let’s first begin by understanding exactly what RNAi is and how it works. 

RNAi is a naturally occurring cellular mechanism that regulates gene expression by silencing messenger RNA (mRNA) after it is transcribed. Rather than modifying DNA, RNAi operates at the RNA level, controlling whether specific genetic instructions are translated into protein. This process is mediated primarily by two classes of RNA molecules: siRNAs and microRNAs (miRNAs). These molecules can essentially be engineered to “hack” the RNAi machinery.  

Both RNAs work by joining a cellular machine called the RNA-induced silencing complex (RISC), which carries out gene silencing. For siRNAs, one strand of the RNA duplex – the “guide” strand – stays in RISC and helps it find a perfectly matching mRNA. Once it finds its target, a protein called Argonaute 2 (AGO2) cuts the mRNA, causing it to break down, preventing the gene from producing its protein. On the other hand, miRNAs work a bit differently; they usually attach less perfectly to their targets and mainly reduce protein production by blocking translation or destabilizing the mRNA, rather than cutting it directly. 

Therapeutic RNAi uses siRNAs to precisely turn off disease-causing genes, including some that traditional drugs cannot target. The success of approved RNAi medicines has proven that this approach works in patients, while also showing that there’s room for improvement.  

The bottlenecks of RNAi-based therapeutics 

New technologies are now beginning to focus on refining siRNA design and how it interacts with the cellular RNAi machinery to make treatments even more potent, longer lasting, and effective in a wider range of tissues. 

This is because, despite the clinical success of RNAi, current therapeutic siRNAs face significant structural limitations that restrict their effectiveness outside the liver. One of the main challenges is ensuring that the correct strand – the antisense or “guide” strand – is efficiently loaded into the RNA-induced silencing complex (RISC). If this strand is poorly selected, gene silencing is weak, which often forces the use of higher doses that could, in turn, increase the risk of side effects. 

Although chemical modifications have improved the stability of siRNAs, helping them resist degradation by nucleases in the body, these modifications can interfere with the RISC machinery, particularly the AGO2 protein that performs the catalytic cleavage of target mRNA. Ultimately, the result is slower or less efficient gene silencing, limiting the potency of siRNA therapies. 

Most current siRNAs use design features like 3’ overhangs or internal chemical modifications, but these do not fully optimize the 5’ end of the antisense strand – the region that is most critical for RISC recognition and guide strand selection. This means that, even with chemical stabilization, the intrinsic activity of the siRNA molecule is not maximized.  

Therefore, developing structural innovations that can enhance this intrinsic potency could enable strong gene silencing at lower doses, opening the door to treating difficult targets, such as aggressive cancers or diseases affecting tissues beyond the liver. 

How the University of North Carolina’s new technology enhances RNAi potency  

Enhanced RNAi Potency

This technology provides a novel platform for enhancing RNAi potency through specific DNA-based modifications at the 5’ end of the antisense strand. By incorporating a nucleotide overhang of two or more deoxythymidines (dT) specifically at the 5’ terminus, the researchers have engineered a “sticky” antisense lead that significantly optimizes the loading and catalytic efficiency of the RISC complex.

 

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This kind of innovation is exactly what UNC-Chapel Hill has produced. With its new technology, it offers a novel platform for enhancing RNAi potency through specific DNA-based modifications at the 5’ end of the antisense strand.  

Essentially, this technology tackles one of RNAi’s core weaknesses by re-engineering the very tip of the siRNA molecule that determines whether gene silencing works efficiently or not. Specifically, the approach introduces a nucleotide overhang of two or more deoxythymidines (dT) at the 5’ terminus. Deoxythymidines are DNA building blocks rather than RNA, and, by adding them as a short overhang, the UNC-Chapel Hill researchers have engineered a “sticky” antisense lead that ultimately improves how efficiently the RISC recognizes, loads, and activates the guide strand. 

In head-to-head studies targeting the well-known cancer-driving genes KRAS and MYC, these 5’ modified dsRNAs showed remarkably superior inhibition across multiple cell lines (including A-431 and MIA-PaCa2) compared to standard commercial modifications. For example, the 2dT and 3dT antisense modifications achieved significantly lower ED50 values (a crucial measure of drug potency, where a lower ED50 indicates higher potency) than standard “Hi2OMe” modified siRNAs. In practical terms, this means that far less of the drug was needed to achieve the same, or better, levels of inhibition, which is a key advantage when safety and tolerability are major concerns. 

Crucially, the platform is broadly applicable rather than gene-specific. Because the deoxythymidine overhang enhances the fundamental interaction between the siRNA and the RNAi machinery, it can be deployed against virtually any gene target, including mutant-specific sequences such as KRAS G12V. Ultimately, by boosting the inherent potency of RNAi, the platform offers a promising new route for expanding RNAi therapies beyond their current boundaries and into more challenging disease areas, such as precision oncology. 

The benefits and real-world applications of UNC’s tech 

Naturally, the primary benefit of UNC-Chapel Hill’s platform is a marked increase in RNAi potency, enabling strong gene silencing at significantly lower doses than conventional chemically modified siRNAs. By improving antisense strand selection and Argonaute-mediated activity, the DNA-based 5′ modifications enhance RISC loading efficiency, which in turn reduces the risk of off-target effects and systemic toxicity. This improved intrinsic activity is particularly valuable for therapeutic applications where dose limitations have constrained RNAi performance. 

The technology also offers high precision and broad flexibility. It has demonstrated mutation-specific silencing of oncogenic targets such as KRAS G12V while sparing wild-type sequences. At the same time, the platform is compatible with existing delivery systems, including lipid nanoparticles and ligand-based approaches, and can integrate seamlessly with established internal chemical modification patterns, making it easy to adopt within current RNAi development pipelines. 

Together, these attributes mean that the platform could expand the potential applications of RNAi across both therapeutic and research settings, as it enables more effective targeting of traditionally “undruggable” oncogenes such as MYC and KRAS, improves the performance of siRNA drug candidates across disease areas, and provides a high-potency gene silencing tool for in vitro and in vivo functional genomics.  

Ultimately, by directly addressing structural bottlenecks at the 5′ end of the antisense strand, this new technology from UNC-Chapel Hill could potentially represent a meaningful advance in siRNA design. Its ability to boost potency, precision, and safety, while remaining compatible with existing RNAi technologies, makes it a promising platform for extending the reach of RNAi therapeutics into more challenging and clinically impactful targets beyond liver-targeted delivery.