Bivalent Display of Dicysteine on Peptide Nucleic Acids for Homogenous DNA/RNA Detection through in Situ Fluorescence Labelling

Fluorogenic probes that signal the presence of specific DNA or RNA sequences are key enabling tools for molecular disease diagnosis and imaging studies. Usually, at least one fluorophore is attached through covalent bonding to an oligonucleotide probe. However, the additional conjugation step increases costs. Here we introduce a method that avoids the requirement for the preparation of fluorescence‐labelled oligonucleotides and provides the opportunity to alter the fluorogenic reporter dye without resynthesis. The method is based on adjacent hybridization of two dicysteine‐containing peptide nucleic acid (PNA) probes to form a bipartite tetracysteine motif that binds profluorescent bisarsenical dyes such as FIAsH, ReAsH or CrAsH. Binding is accompanied by strong increases in fluorescence emission (with response factors of up to 80‐fold and high brightness up to 50 mL mol^?1 cm^?1). The detection system provides sub‐nanomolar limits of detection and allows discrimination of single nucleotide variations through more than 20‐fold changes in fluorescence intensity. To demonstrate its usefulness, the FIAsH‐based readout of the bivalent CysCys‐PNA display was interfaced with a rolling‐circle amplification (RCA) assay used to detect disease‐associated microRNA let‐7a.