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.

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