Circular RNAs (circRNAs) are a unique class of RNA molecules found in nearly all eukaryotic organisms. Unlike traditional linear RNAs, circRNAs form covalently closed loops through a process called backsplicing, where a segment of RNA is joined end-to-end to create a continuous circular structure. Because their sequence does not align in a straightforward manner with the genome, circRNAs remained undetected for years, even as RNA sequencing technologies rapidly evolved. However, research in the past decade has revealed the existence of over 3 million unique circRNAs, many of which appear to have specific biological roles.

Researchers from Flinders University discuss the latest strategies for detecting and quantifying these elusive RNA molecules. The authors highlight several challenges in studying circRNAs, including their typically low expression levels and the difficulty in accurately determining their full sequence from short-read sequencing data. To overcome these hurdles, researchers are developing more advanced sequencing technologies, such as long-read sequencing and bioinformatics pipelines specifically designed to enhance circRNA detection.

CircRNA enrichment strategies preceding next-generation sequencing

As distinct from mRNA-seq where polyadenylated (poly(A)+ RNA) is enriched, circRNAs are enriched from total RNA is subjected to linear RNA removal via ribosomal RNA (rRNA) depletion or depletion of poly(A)+ RNA (poly(A)− pool) followed by RNase R treatment. Induro-RT-mediated circRNA enrichment is achieved without RNase R treatment through rolling circle amplification, followed by bead purification to enrich long concatemeric cDNA produced from circRNAs from shorter linear RNAs.

In addition to sequencing advancements, scientists are also working on laboratory-based techniques to visualize and quantify circRNAs directly in cells. These new methods are crucial for understanding the biological functions of circRNAs and their potential roles in diseases such as cancer and neurodegenerative disorders. As technology continues to advance, researchers will be better equipped to fully characterize circRNA variants and explore their significance in gene regulation and cellular processes.