Duplex-Specific Nuclease (DSN), originating from the Paralithodes camtschaticus, exhibits sequence-independent DNA cleaving activity while remaining inactive toward RNA. This special property of DSN offers potential in detection of ribosomal RNA (rRNA), the most abundant molecule in active cells and an attractive target for environmental pathogenic biosensors, using DNA:RNA hybridization. Targeting the V3 hypervariable region of E. coli 16S rRNA, we designed and tested a DNA probe to specifically capture 16S rRNA. The hybridization efficiency of 16S rRNA with the DNA probe and the specificity of DSN recognition and cleaving, are time and temperature dependent. Using a DNA probe labeled with a fluorophore, we observe the accumulation of fluorophores over time as an indication of DSN activity and recycling of intact RNA. The signal amplification factor is defined as the ratio of signal collected following DSN digestion to signal of hybridized duplexes prior to DSN addition. Additionally, DSN’s ability to discriminate between non-complementary sequences is tested using mismatched probes. This assay achieves highly specific detection of E. coli using native rRNA, demonstrating RNA viability for pathogen detection in environmental samples.

Detection of E. coli 16S ribosomal RNA using duplex specific nuclease-mediated target recycling signal amplification

2019 Fall National Meeting

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