Genetically Encoded Biosensor Platform for Real-Time Detection of 2-5A and Double-Stranded RNA in Live Cells

Background

The detection and quantification of immune signaling molecules such as 2’-5’-linked oligoadenylates (2-5A) and double-stranded RNA (dsRNA) are critical in the fields of immunology, virology, and clinical diagnostics. These molecules play pivotal roles in the innate immune response, particularly in the activation of RNase L, which is involved in antiviral defense, apoptosis, and regulation of cell proliferation. Accurate measurement of 2-5A and dsRNA levels is essential for understanding immune activation during infections, autoimmune diseases, and for monitoring the efficacy of therapeutic interventions. Furthermore, the ability to monitor these molecules in real time and in live cells is increasingly important for drug discovery, especially for high-throughput screening of compounds that modulate immune pathways.

Current approaches for detecting 2-5A and dsRNA suffer from significant limitations. Most existing assays are endpoint measurements that require cell lysis or destruction of the sample, preventing longitudinal studies in the same cells or tissues. These methods are often labor-intensive, time-consuming, and not amenable to high-throughput formats, which restricts their utility in large-scale drug screening or clinical diagnostics. Additionally, traditional assays may lack the sensitivity or specificity needed to detect low-abundance signaling molecules in complex biological samples. The inability to perform real-time, non-destructive, and dynamic monitoring of these immune messengers hampers both basic research and the development of new therapeutics targeting the 2-5A/RNase L pathway.

Technology

This technology is a genetically engineered biosensor designed for real-time, non-destructive detection of the interferon-induced second messenger 2’-5’-linked oligoadenylates (2-5A) and double-stranded RNA (dsRNA) in live cells, lysates, or tissues. The core of the system is a fusion protein that incorporates RNase L (or its functional ANK domain) flanked by split fragments of a reporter protein, such as luciferase or a fluorescent protein (e.g., GFP, sfCHERRY), connected by flexible peptide linkers. Upon binding of 2-5A to the RNase L domain, the protein dimerizes, bringing the split reporter fragments together and reconstituting their activity, which generates a detectable luminescent or fluorescent signal. The construct can be further tailored with nuclear localization or export signals for subcellular targeting and is compatible with various expression systems and sample types, making it suitable for high-throughput applications in immune monitoring, infection diagnostics, and drug screening.

This solution is differentiated by its ability to provide live, real-time, and high-throughput measurement of 2-5A and dsRNA dynamics, overcoming the limitations of traditional endpoint and destructive assays. Its modular design allows for the use of different reporter proteins and subcellular targeting, while the sensitivity and specificity are demonstrated by its nanomolar detection range and lack of response to 2-5A when key binding mutations are introduced. The system’s versatility is further enhanced by its compatibility with multiple detection modalities and sample types, as well as its capacity for stable cell line generation for repeated assays. By enabling dynamic monitoring of innate immune signaling, this biosensor platform opens new avenues for research, diagnostics, and therapeutic development in immunology, virology, and related fields.

Benefits

• Enables real-time, non-destructive detection of 2-5A and double-stranded RNA in live cells and tissues
• High sensitivity and specificity for nanomolar concentrations of 2-5A
• Compatible with multiple reporter proteins (luciferase, GFP, sfCHERRY) and detection modalities (luminescence, fluorescence)
• Modular design allows subcellular targeting and customization via localization signals and mutations
• Suitable for high-throughput screening of small molecule modulators of the 2-5A/RNase L pathway
• Applicable to diverse sample types including live cells, lysates, bodily fluids, and tissues
• Facilitates monitoring of immune responses, viral infections, and therapeutic efficacy in real time
• Enables creation of stable reporter cell lines for repeated and scalable assays

Applications

• Live-cell immune response monitoring
• High-throughput antiviral drug screening
• Real-time infection diagnostics
• Therapeutic efficacy assessment
• Autoimmune disease biomarker detection

Additional info

This biosensor features a fusion protein containing an RNase L sequence flanked by split reporter fragments via flexible linkers. Upon binding 2’-5’-linked oligoadenylates, the protein dimerizes, reconstituting the reporter fragments to restore functional activity. This mechanism produces detectable luminescence or fluorescence, allowing real-time, non-destructive quantification of 2-5A and double-stranded RNA in live cells or tissues for high-throughput immune monitoring and drug screening.