We have developed a single probe method for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by polymerases. An abasic peptide nucleic acid (PNA) probe—containing a reactive amine instead of a nucleotide at a specific position in the sequence—for detecting a microRNA was conjugated to superparamagnetic beads. These beads were incubated with a sample containing microRNA, a biotinylated reactive nucleobase—containing an aldehyde group—that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. Single molecules of the biotin-labeled probe were then labeled with streptavidin-β-galactosidase (SβG), the beads were resuspended in a fluorogenic enzyme substrate, loaded into an array of femtoliter wells, and sealed with oil. The array was imaged fluorescently to determine which beads were associated with single enzymes, and the average number of enzymes per bead was determined. The assay had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mis-match. This assay was used to measure microRNA-122 (miR-122)—an established biomarker of liver toxicity—extracted from the serum of patients who had acute liver injury due to acetaminophen, and control healthy patients. All patients with liver injury had higher levels of miR-122 in their serum compared to controls, and the concentrations measured correlated well with those determined using RT-qPCR. This approach allows rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for clinical use.

Circulating microRNAs are biomarkers reported to be stable and translational across species. MicroRNA-122 (miR-122) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). We developed a single molecule, dynamic chemical labeling (DCL) assay to directly detect miR-122 in blood. The DCL assay specifically measured miR-122 directly from 10 μL of serum or plasma without any extraction steps, with a limit of detection of 1.32 pM that enabled the identification of DILI. Testing of 192 human serum samples showed that DCL accurately identified patients at risk of DILI after acetaminophen overdose (area under ROC curve 0.98 (95% CI; 0.96–1), P < 0.0001). The DCL assay also identified liver injury in rats and dogs. The use of specific captured beads had the additional benefit of stabilizing miR-122 after sample collection, with no signal loss after 14 days at room temperature, in contrast to PCR that showed significant loss of signal. RNA sequencing demonstrated the presence of multiple miR-122 isomiRs in the serum of patients with DILI that were at low concentration or not present in healthy individuals. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analyzing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. We conclude that the DCL assay can accurately measure miR-122 to diagnose liver injury in humans and other species and can overcome microRNA stability and isomiR challenges.

Over the last decade, circulating microRNAs have received attention as diagnostic and prognostic biomarkers. In particular, microRNA122 has been demonstrated to be an early and more sensitive indicator of drug-induced liver injury than the widely used biomarkers such as alanine aminotransferase and aspartate aminotransferase. Recently, microRNA122 has been used in vitro to assess the cellular toxicity of new drugs and as a biomarker for the development of a rapid test for drug overdose/liver damage. In this proof-of-concept study, we report a PCR-free and label-free detection method that has a limit of detection (3 standard deviations) of 15 fmoles of microRNA122, by integrating a dynamic chemical approach for "Single Nucleobase Labelling" with a bead-based platform (Luminex) thereby, in principle, demonstrating the exciting prospect of rapid and accurate profiling of any microRNAs related to diseases and toxicology.