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

Abstract

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 tC^o_Azo 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/tC^o_Azo-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 tC^o_Azo residues provided a concentration-dependent signal. The results indicate that tC^o_Azo is a promising tool for fine tuning MB properties for fluorescent- and SERS-based diagnostic applications.