Fluorogenic oligonucleotide cleavage probes with a branched linker improve RNA detection

Area of Science:

• Molecular Biology
• Biochemistry
• Oligonucleotide Chemistry

Background:

• Quantitative polymerase chain reaction (qPCR) relies on fluorescent probes for DNA/RNA quantification.
• Fluorogenic probe performance is critically dependent on fluorophore, quencher, and linker design.
• TaqMan® probes are widely used in research and clinical diagnostics.

Purpose of the Study:

• To develop a highly modular linker for fluorogenic oligonucleotide probes.
• To create a multiplexed assay for simultaneous detection of multiple RNA viruses.
• To compare the performance of a novel 'X' linker with existing linker strategies.

Main Methods:

• Design and synthesis of a three-way branched glycerol 'X' linker for TaqMan® probes.
• Incorporation of various fluorophores (Flu, Hex, Cy5, Cy5.5) and quenchers (BHQ1, BHQ2).
• Development of a four-color reverse transcription qPCR (RT-qPCR) 'tetraplex' assay for viral RNA detection.

Main Results:

• The 'X' linker enabled a tetraplex RT-qPCR assay for SARS-CoV-2, influenza A, and influenza B viruses.
• Probes with the 'X' linker achieved single-molecule detection limits near four copies.
• The 'X' linker demonstrated superior performance over an arabinoside-based (ara) linker, showing distinct cleavage mechanisms during primer extension.

Conclusions:

• The glycerol 'X' linker is a highly effective and modular component for fluorogenic probes.
• Linker structure significantly impacts oligonucleotide probe performance in qPCR assays.
• This linker facilitates the development of sensitive and multiplexed diagnostic tools for infectious diseases.