Cytosine mimic azophenoxazine tCOAzo quenches FAM fluorescence and provides concentration-dependent Raman quenching upon molecular beacon hybridization

The molecular beacon (MB) strategy finds diverse applications in bioimaging, biosensing and disorders diagnostics. Classic MBs are stem-loop oligonucleotides modified with a reporter and a quencher at the 5′- and 3′- ends. Hybridization with a specific target leads to spatial separation of the quencher and the reporter with subsequent changes in spectral signal, for example, an increase in reporter fluorescence emission. However, hydrophobic interactions between a conventional bulky quencher and the reporter might influence hybridization properties and limit stem length in MB design. In this work, we studied the synthetic nucleobase analog tCoAzo mimicking cytosine, capable of quenching reporter (FAM) fluorescence in folded MBs while minimally affecting their tharmal stability compared to MBs with classic FAM/BHQ1 labels. In addition, we conducted a preliminary assessment of the applicability of FAM/tCoAzo-modified MBs for SERS-based techniques using short single-stranded and long double-stranded DNA fragments from Fusarium avenaceum elongation factor 1a DNA as matrices. SERS intensity was decreased in the presence of matrix DNA compared to the scrambled sequence; and the MB modified with three tCoAzo residues provided a concentration-dependent signal. The results indicate that tCoAzo is a promising tool for fine tuning MB properties for fluorescent- and SERS-based diagnostic applications. © 2025 Elsevier B.V., All rights reserved.