Biology of Circular RNAs and Methodological Approaches to Their Study Michaela Ruckova, Dagmar Al Tukmachi & Ondrej Slaby Chapter First Online: 04 November 2023 232 Accesses Part of the RNA Technologies book series (RNATECHN,volume 14) Abstract From initially overlooked as peculiarities of uncertain biological importance or rare isoforms generated as a result of splicing errors to commonly regarded as relevant regulatory molecules, circular RNAs (circRNAs) now represent an extensively studied subgroup of non-coding RNAs (ncRNAs). The structural uniqueness of these endogenous biomolecules confers resistance to exonuclease activity, resulting in higher stability than observed in their linear counterparts, which alongside their reported function of gene expression regulators, predisposes them to potential use as robust biomarkers and/or powerful therapeutic targets. During the last decade, we have witnessed a boom in circRNA research deciphering various mechanisms of their biogenesis and an in-depth description of their biological functions, including miRNA and protein sponging, yet regulation of these events remains unclear. Despite recent advances in high-throughput genomic technology and novel bioinformatic approaches allowing circRNAs characterization in detail, analyzing these molecules continues to be challenging, leaving multiple biological knowledge gaps unsolved.