Aptamer-Based Fluorescent Assays for Detection of the Host-Response Viral Biomarker 3′-Deoxy-3′,4′-Didehydro-Cytidine

Abstract Improved viral diagnostics are urgently needed to strengthen pandemic preparedness and combat antimicrobial resistance, highlighting the importance of 3′-deoxy-3′,4′-didehydro-cytidine (ddhC), a host-derived, pan-viral biomarker that addresses key limitations of PCR-based diagnostics. Current methods for ddhC measurement rely on liquid chromatography-mass spectrometry (LC−MS) or nuclear magnetic resonance spectroscopy, which are accurate but costly and impractical for rapid or point-of-care testing. Here, we report the development of aptamer-based fluorescent assays for selective ddhC detection. Low-micromolar-affinity DNA aptamers were isolated using capture-systematic evolution of ligands by exponential enrichment (SELEX) and characterised by isothermal titration calorimetry. Using a common core aptamer sequence, three fluorescence assay formats−strand displacement, molecular beacon, and dye displacement−were developed and evaluated. Independent LC−MS analysis demonstrated a strong correlation between creatinine-normalised urinary and serum ddhC concentrations in inpatient samples (R = 0.98, p <0.0001), supporting urine as a non-invasive matrix for ddhC detection. Among the three aptamer-based formats tested, the dye displacement assay exhibited the lowest functional Kd but was limited in urine by inner filter effects, whereas a Cy5-labelled strand displacement assay overcame this limitation and detected ddhC in patient urine with moderate correlation to LC−MS (R = 0.76, p = 0.001). Together, these results establish aptamer-based sensing approaches for ddhC and demonstrate their feasibility in clinical urine samples, providing a foundation for further optimisation toward accessible, non-invasive, and rapid detection of acute viral infection.