Sensitive Point-of-Care miRNA Detection Using a One-Pot, Isothermal Assay

Rapid, RNA Extraction-Free Detection of miRNAs Biomarkers for Accurate Cancer Diagnosis and Prognosis

This CRISPR-Cas12a-based assay allows for the rapid, sensitive detection of microRNAs using a one-pot, isothermal process. MicroRNAs(miRNAs) are endogenous and short, non-coding single-stranded RNAs. They participate in various biological functions such as cell proliferation, differentiation, and cell death. The dysregulated expression of miRNA links to the development of different diseases, like cancer. miRNAs are found in bodily fluids, making the detection of circulating miRNAs a promising and effective strategy in liquid biopsy for cancer diagnosis, prognosis, and monitoring. However, identifying miRNA sequences is complicated by their short length and the high likelihood they closely resemble sequences from other miRNA families. While many RNA detection protocols exist, they suffer from limited practicality due to complicated workflows, the need for specialized equipment, low sensitivity, and long turnaround times.

Researchers at the University of Florida have developed EXTRA-CRISPR, a one-pot, isothermal assay for sensitively detecting miRNAs. This assay combines off-the-shelf reagents into a single reaction for fast, sensitive, and specific detection of miRNAs while reducing the chance of contamination and eliminating the need for specialized equipment.

Application

Detection of miRNA biomarkers for point-of-care cancer diagnosis and prognosis

Advantages

• Achieves turnaround in as few as 20 minutes using typical laboratory equipment, allowing point-of-care applications
• Exponential amplification unlocks high sensitivity and a wide range of detection, yielding results comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) tests
• Implements detection as a one-pot assay, reducing the risk of contamination
• Achieves single-nucleotide specificity, enabling distinction between miRNA species with high sequence similarity
• Eliminates the need for a protospacer adjacent motif (PAM), allowing for greater flexibility when designing padlock probes
• The cost of goods (COG) per reaction is $0.60 at the research scale, highlighting its affordability and potential for scalable production

Technology

The core of EXTRA-CRISPR is a miRNA-specific padlock probe that detects weakly concentrated miRNA by rolling circle amplification (RCA). This padlock probe consists of single-stranded DNA with a part of its sequence complementary to the target RNA for specific identification. The probe forms a circular template around which a polymerase extends the miRNA continuously to copy the template sequence repetitively, creating a long DNA strand. Throughout these steps, EXTRA-CRISPR deploys various enzymes, including ligase, polymerase, and Cas12a, to amplify the amount of miRNA, facilitating the detection of miRNA biomarkers.

Ligase transforms the padlock probe into a circular template, with miRNA serving as a splint. Polymerase enables RCA, and the cis-cleavage activity of Cas12a cuts the RCA product, generating cleaved segments that exponentially trigger secondary amplification.

At the same time, Cas12a can cleave a fluorescent reporter using its trans-cleavage activity, generating a fluorescent signal detectable in real-time by standard quantitative polymerase chain reaction tools or even with smartphone applications. This streamlined process of miRNA detection enhances the feasibility of using miRNAs for point-of-care diagnostics.