In recent years, the advent of high-throughput RNA-sequencing technology has brought attention to a newly described RNA species, circular RNA (circRNA). Unlike traditional linear RNA, circRNA forms a 3'-5' covalently closed ring, exhibiting structural stability without the need for 5'-cap or 3'-poly(A) tails. With diverse functions and widespread presence in various cells, circRNAs, particularly exonic circRNAs (ecircRNAs), have gained prominence as regulators of gene expression. Notably, their role as microRNA sponges and involvement in diseases like cancer has spurred interest. Amid these discoveries, the unique structural advantage of circRNAs, offering enhanced stability against exonucleases, has positioned them as potential candidates for RNA vaccine development, particularly with the rise of RNA vaccines. There is a growing need to mimic circRNA biology by synthesizing tailored circRNAs with specific properties. We summarize current strategies for producing circRNAs in vitro and in vivo.
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Introduction of circRNAs
The concept of circRNAs dates back to 1976 when Sanger et al. identified covalently closed circRNA molecules in viroids. Over the years, circRNAs were discovered in various organisms, including yeast and the hepatitis delta virus. However, their significance remained largely overlooked until the advent of high-throughput sequencing in 2012, when Salzman et al. revealed the abundance of circRNAs in human cells. This discovery sparked renewed interest, leading to the exploration of circRNA functions, such as acting as microRNA sponges and exhibiting greater stability than linear mRNAs.
Fig. 1 Timeline of circular RNA (Chen X., Lu Y. 2021).
In vitro circRNA synthesis
circRNAs (circular RNAs) are synthesized in vitro through a multi-step process involving the generation of a linear RNA precursor and subsequent circularization. Various methods, including chemical and enzymatic approaches, are employed for both the synthesis of linear RNA and the subsequent ligation to form a covalently closed loop.
Linear RNA Precursor Synthesis
Chemical Strategies: Using synthesizer machines, chemical synthesis involves the use of phosphoramidites, derivatives of nucleotide triphosphates, to build linear oligonucleotides. Protective groups are used to prevent undesired reactions, ensuring high purity. However, this method is limited to shorter RNA molecules.
Enzymatic Strategies: In vitro transcription (IVT) using phage RNA polymerases (T7, SP6, or T3) is a common enzymatic approach. DNA templates with cognate promoters are transcribed to produce longer linear RNAs, overcoming the length limitations of chemical synthesis. Strategies are employed to address issues such as the first nucleotide requirement and the run-off nature of polymerases.
Ligation of Linear RNA Precursor
Chemical Strategies: Chemical methods involve intramolecular reactions between the first and last nucleotides of the linear RNA, using reagents like cyanogen bromide or EDC to activate circularization. Alternative reactive groups can be introduced for efficient bond formation. However, non-natural linkages may impact circRNA structure and function.
Enzymatic Strategies: DNA and RNA ligases (T4 bacteriophage-derived) are commonly used for enzymatic ligation. Strategies like using a short DNA oligonucleotide splint or adding helper oligonucleotides are employed to facilitate efficient circularization. Ligases exhibit substrate specificity, and the choice of ligase impacts efficiency.
In vitro circRNA Synthesis using Ribozymes
Group I Introns (PIE System): Adapted from naturally occurring introns, the permuted intron-exon (PIE) system involves the autocatalytic activity of group I introns to splice themselves, leading to circularization. The choice of group I intron affects circularization efficiency, and a bioinformatics pipeline aids in predicting optimal permutations.
Group II Introns: Similar to group I introns but with a different mechanism, group II introns, when permuted, can also facilitate circRNA synthesis. Unlike group I introns, group II introns allow for the production of a perfect sequence mimic without retaining terminal portions of native exons.
Ribozymes from Subviral Agents: Ribozymatic elements from subviral agents, such as hairpin ribozymes, can be used for directed circRNA synthesis in vitro. This approach, while generating small circRNAs with high sequence homogeneity, has drawbacks, including the retention of ribozymatic sequences in the final circRNA.
In vivo circRNA synthesis
CircRNAs, synthesized in vivo, involve the overexpression of a plasmid carrying a minigene with a naturally circularized sequence post-transcription. Typically, circRNA minigenes encompass an exonic region for circularization, along with 5' and 3' flanking intronic sequences containing splicing motifs. Four minigene categories include (i) introns with complementary sequences, (ii) those with binding motifs for regulatory proteins, (iii) those with ribozymatic activity, and (iv) introns from metazoan tRNAs. Notably, circRNAs from plasmids are favored for endogenous circRNA models due to in situ generation.
The ICS strategy, divided into repetitive (e.g., Alu elements) and non-repetitive categories, relies on endogenous introns containing complementary sequences. Repetitive ICS, often derived from Alu elements in humans, efficiently drives backsplicing. Non-repetitive ICS, such as those in the Sry gene, involve endogenous introns with non-repetitive complementary sequences.
The minigene system using RNA binding protein (RBP) motifs incorporates binding sites for RBPs like Muscleblind and Quaking, enhancing backsplicing. The Muscleblind-based system utilizes introns from the Mbl gene, promoting backsplicing through RBP dimerization. The Quaking-based system involves direct binding of QKI to the reporter pre-mRNA, facilitating circRNA biogenesis.
The permuted group I intron-exon (PIE) system, an in vitro strategy, is unique for in vivo circRNA production. It employs plasmids containing permuted group I intron templates, producing linear transcripts that circularize through self-splicing ribozymatic activity. The PIE method, effective in bacteria and fungi, was initially unclear for mammalian cells due to immunogenicity concerns.
Additionally, the tRNA intron-based method exploits metazoan tRNA splicing, generating circRNAs by cleaving intron-containing pre-tRNAs and ligating the excised introns. The Tornado system, an enhanced version, efficiently produces circRNAs in human and Drosophila cells. This method is versatile, producing functional circRNA aptamers with applications in gene regulation and molecular targeting.
In conclusion, the diverse functions and structural stability of circRNAs, highlight their significance as regulators of gene expression. The synthesis methods discussed above, both in vitro and in vivo, offer valuable tools for studying circRNA biology and pave the way for potential applications, including the development of circRNA-based therapeutics and RNA vaccines in the evolving landscape of RNA technology.
References
- Chen X., Lu Y. Circular RNA: Biosynthesis in vitro. Frontiers in Bioengineering and Biotechnology. 2021, 9: 787881.
- Obi P., Chen Y. G. The design and synthesis of circular RNAs. Methods. 2021, 196: 85-103.
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