Background

Circular RNAs (circRNAs) are unique RNAs characterized by their covalently closed-loop structures. These structures confer increased stability compared to their linear counterparts by rendering them resistant to exonuclease-mediated degradation [1]. This natural stability makes circRNAs promising candidates for RNA-based therapeutics, including gene regulation, biomarker development, and targeted drug delivery [2]. As the diversity of circRNA design strategies and applications continues to expand, accurately assessing circRNA yield and purity has become an emerging and critical need.

Current circRNA detection methods [3] mainly detect and quantify a relatively small amount of circRNAs in a specific context. High-throughput RNA sequencing (RNA-Seq), combined with bioinformatics, analyzes various types of genome-wide circRNAs present in low abundance [4]. By targeting the junction sequences of RNA back-splicing/ligation, quantitative reverse transcription PCR (qRT-PCR) offers sensitive and specific quantification for trace circRNA from an RNA pool [5]. Even though these approaches effectively detect circRNA, none directly assess the accompanying linear RNAs in the same pool. This is especially crucial for quality control and downstream applications of circRNA after mass production.

Linear RNA contaminants can be found in a circRNA preparation for several reasons, such as yet-to-be circularized RNA, somehow disconnected circRNAs, or any byproduct RNAs with free 3' ends in circRNA production processes. Here, we present a streamlining assay that leverages the activity of template-independent RNA polymerase to append adenine tails (polyA) to the 3' ends of linear RNAs [6]. As a consequence, the prerequisite for the success of this polyA tailing assay significantly relies on optimizing denaturing formaldehyde gel conditions and testing appropriate tailing assay parameters in-house. Denaturing gel electrophoresis offers better resolution and prevents RNA degradation, reducing misleading band shifts compared to non-denaturing TBE gels. This strategy clearly tells that any linear RNA remains in the circRNA products, allowing for a reliable assessment of circRNA purity, predominantly when the sample is mainly composed of circRNA.

Exonuclease resistance is a circRNA signature, distinguishing it from linear RNA [7]. However, RNase R can be used for linear RNA removal in circRNA preparation rather than for detecting circRNA. To evaluate linear RNA in the bulk of circRNA preparation more straightforwardly and intuitively, we repurposed the polyA tailing assay from its primary application for in vitro mRNA preparation [8]. This method provides an effective and practical approach to assessing circRNA integrity.