First Single Molecule Counting (SMC™) Application with microRNAs by a PCR Free Method

Circulating microRNAs (miRNAs) are a group of small RNA molecules that have been shown to be involved in several human diseases, such as cancers, neurodegenerative disease, and inflammation. Their sequences are highly conserved across species. miRNAs are present in biological fluids, are differentially expressed and can be used as biomarkers reported to be translational for research and drug development as improved diagnostic assays. Multiple techniques used to measure miRNA expression in biological fluids are labor-intensive and subject to variability. The objective of this study was to develop an extraction-free and amplification-free detection method using Single Molecule Counting (SMC™) technology and the SMCxPRO™ instrument combined with dynamic chemical labelling (DCL) chemistry by DESTINA for the direct detection of microRNAs in biological samples. As a proof of concept, miR-122 (miR-122-5p), a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI) has been selected. A DESTINA abasic probe for detecting miR-122 is conjugated to superparamagnetic beads. These beads are added to DILI patient serums treated with DESTINA Stabiltech and  incubated with a biotinylated DESTINA SMART Nucleobase. Following miR-122 target molecule hybridization to the DESTINA probe, the reactive SMART nucleobase is then covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. The biotin-labeled superparamagnetic beads are then incubated with Alexa Fluor®647-conjugated streptavidin and the labeled heteroduplex complex is cleaved from the superparamagnetic beads before being detected by the SMCxPRO™instrument. This is the first example where SMC™ technology has been used for molecular applications, allowing rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for research.