Nucleic acid-based assays allow for rapid detection of bacterial and viral pathogens and sequence-specific analysis of their genetic signatures. In this respect, hybridization probes are important tools to ensure specificity of the analysis. Fluorescent light up aptamers (FLAPs), which bind low-fluorescent dye ligands and increase their fluorescence, offer an advantage of label-free signal reporting. Therefore, FLAP-based hybridization probes are promising for molecular diagnostics.<br/>Recently, a dapoxyl dye-binding FLAP has been reported. We have demonstrated that the same aptamer sequence can also bind and enhance fluorescence of other fluorogenic dyes including commercially available arylmethane dyes. Such binding promiscuity allows one to design an aptasensor with multiwavelength reporting capabilities depending on the dye used. We have designed a split dapoxyl aptamer (SDA) probe and applied it to the detection of the amplified fragments of bacterial and/or viral genomes obtained using nucleic acid sequence-based amplification (NASBA). Four different fluorogenic dyes were explored with the SDA probe, which can be designed do differentiate the nucleic acid targets down to single-nucleotide substitutions. The linear dynamic range and limit of detection are not significantly affected by the changes in the dye used. The signal can be conveniently observed by the unaided eye when excited with a portable light source. The system is tolerant to potential inhibitors from the amplification reaction, so that the amplicon can be interrogated by the probe directly in the amplification sample without the need of the amplicon isolation. This opens a possibility to use the SDA probe in a one-tube format, where all the components for the amplification and detection reactions are added simultaneously. We have also discovered that nucleotide substitutions at specific positions of the dye-binding core of the aptamer affect its ability to trigger fluorescence of some dyes, but not the others. This property can be used to design a set of aptasensors for multiplex target analysis using one and the same aptamer as a scaffold.

Split fluorescent light-up aptamer probes for label-free pathogen detection

ACS Fall 2021

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