Rapid miRNA Imaging in Cells Using Fluorogenic Templated Staudinger Reaction Between PNA-Based Probes

Reactions templated by a specific nucleic acid sequence have emerged as an attractive strategy for nucleic acid sensing. TheStaudinger reaction using an azide-quenched fluorophore and a phosphine is particularly well suited by virtue of its bioorthogonalityand biocompatibility. The reaction is promoted by a complementary nucleic acid that aligns the phosphine with the azide-quenchedfluorophore. Cellular RNAs can catalyze the Staudinger reaction and signal amplification can be achieved through multipleturnover of the template. Peptide nucleic acids (PNA) provide a convenient platform for the preparation of specific probesas they combine desirable hybridization properties, robust synthesis, ease of fluorophore conjugation, and high biochemicalstability. Herein, we describe protocols for fast fluorescent detection of miRNAs in human cells with PNA-based probes viareductive unquenching of bis-azidorhodamine by trialkylphosphine.