Fluorogenic oligonucleotide cleavage probes with a branched linker improve RNA detection.

Fluorescent probe-based quantitative polymerase chain reaction (qPCR) is essential for DNA/RNA quantification widely used in research and clinical diagnostics. The performance of fluorogenic probes depends heavily on their design, particularly the identities of the fluorophore and quencher moieties, and the linkers used to attach them to oligonucleotides. Here we report a highly modular, three-way branched glycerol 'X' linker in fluorogenic TaqMan® type oligonucleotide probes for multiplexed, reverse transcription qPCR (RT-qPCR). The flexible 'X' linker served as an internal attachment point for various quenchers (BHQ1, BHQ2) in probes containing a variable fluorophore at the 5' end (Flu, Hex, Cy5, Cy5.5). A four-color RT-qPCR 'tetraplex' assay was thereby developed for distinguishing between RNA genomes from SARS-CoV-2, influenza A, and influenza B viruses in a single reaction. The 'X' linker exhibited superior performance with single-molecule detection limits approaching four copies, compared to an internal arabinoside-based (ara) linker strategy, demonstrating the presence of competing processes during primer extension, one where Taq exonuclease activity cleaves the fluorogenic X probe leading to productive fluorescence, and the second where the ara probe is displaced from the PCR template without cleavage. Together these results demonstrate the importance of linker structure selection in oligonucleotides for developing highly effective fluorogenic probes for qPCR.