Wash-free FISH of bacterial ribosomal RNAs by benzo[a]pyrene-modified oligonucleotides

Abstract

Rapid and reliable bacterial detection methods are essential for appropriate antibiotic treatment of infectious diseases. Fluorescence in situ hybridization (FISH) is one of the most promising detection methods for the culture-independent detection of bacteria. However, standard FISH assays involve stringent washing procedures that result in poor reproducibility of assay results. Therefore, reliable FISH assays require fluorescent oligonucleotides that produce strong fluorescence in the presence of complementary RNAs. Here, we report the synthesis and photophysical properties of OMCbpy fluorescent oligonucleotides, which contain a 2′-O-benzo[a]pyrenylmethylcytidine (Cbpy) unit in the middle of each 2′-O-methyl RNA sequence. Fluorescence studies showed that the fluorescence emission of OMCbpy was enhanced up to 39-fold in the presence of complementary RNAs under slightly acidic conditions. Fluorescence microscopy analysis revealed that wash-free FISH with OMCbpy allows for detection of 16S ribosomal RNAs in fixed Escherichia coli (E. coli) cells with high reproducibility.