Circular RNA (circRNA) is a unique type of noncoding RNA molecule. Compared with traditional linear RNA, circRNA is a covalently closed circle produced by a process called backsplicing. CircRNA is abundant in many cells and has rich functions in cells, such as acting as miRNA sponge, protein sponge, protein scaffold, and mRNA regulator. With the continuous development of circRNA study, circRNA has also played an important role in medical applications, including circRNA vaccines and gene therapy. The in vitro preparation of circRNAs is naturally indispensable for the in-depth study of circRNAs to further validate their biological functions, of which in vitro synthesis is a necessary process.

A Brief History of circRNAs

The concept of circRNAs was proposed in 1976. Extensive research on circRNAs began in 2012 with the discovery of large amounts of circRNAs in human cells. These researches have improved yields of circRNAs, realized the synthesis of large circRNA molecules, and expanded the applications of circRNAs.

Fig. 1 Timeline of circular RNA. (Chen, X. et al., 2021)

With the research fever of scholars on circRNA, the in vitro preparation methods of circRNA have been gradually developed. For the past few years, researchers have achieved highly efficient expression of circRNAs in cells using autocatalytic transcriptsor viroid scaffolds. At the same time, researchers had also developed several methods for the synthesis of circRNAs in vitro, such as chemical method, enzymatic method, and ribozyme method. These methods produced circRNAs by ligating the ends of linear RNA precursor.

Fig. 2 Schematic diagram of circRNA synthesis in vivo and in vitro.

Synthesis of circRNAs In Vitro

Using linear RNA as a precursor, the current common idea for in vitro synthesis of circRNA is to join the two ends to form a covalently closed loop structure. Researchers have developed several methods for linear RNA precursor ligation in vitro. These ligation methods include chemical ligation, enzymatic ligation, and ribozyme method.

Chemical Ligation

Chemical ligation is realized by using cyanogen bromide (BrCN) or 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide to link DNA-RNA hybrids. However, this method suffers from low ligating efficiency and biosafety concerns. In addition, chemical ligation forms 2′, 5′-phosphodiester bonds instead of the natural 3′, 5′-phosphodiester bonds. Therefore, chemical ligation is not a common ligation method. Researchers are more interested in the biosynthesis of circRNAs, which includes enzymatic ligation and ribozyme method.

Enzymatic Ligation

Enzymatic ligations are realized by catalytic reactions of several enzymes from the bacteriophage T4, including T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2).

Fig. 3 Strategies for enzymatic ligations. (Chen, X. et al., 2021)

• T4 Dnl

Using ATP as a cofactor, nicks in double-stranded DNA, double-stranded RNA, or DNA-RNA hybrid strands can be ligated. In cyclization experiments of single-stranded RNA, DNA splints (splints) are usually designed with sequences complementarily paired with the ends of the RNA nick to create a localized hybrid double strand, which can be recognized by T4 DNA ligase 1, which connects the 5' end to the 3' end to form a phosphodiester bond, resulting in the cyclized RNA.Due to the T4 DNA ligase I has a low turnover efficiency on RNA substrates, a large amount of the enzyme is required to achieve RNA ligation.

• T4 Rnl 1

T4 Rnl 1 is a more common ligase for RNA ligation. T4 Rnl 1 catalyzes the nucleophilic attack of the 3′-OH terminus onto the activated 5′-terminus to form a covalent 5′, 3′-phosphodiester bond and produces circRNAs. It is worth noting that T4 Rnl 1 has different preferences for the nucleotides of the 5′-terminus and 3′-terminus: A > G ≥ C > U for the 3′-terminal nucleotide acceptor, and pC > pU > pA > pG for the 5′-terminal nucleotide donor. In addition to the auxiliary role of RNA secondary structure, RNA splints can also be designed to bring the ends closer together, requiring the design of incompletely complementary splint sequences that remain single-stranded at 2-3 nucleotides at both ends.

• T4 Rnl 2

T4 Rnl 2 can also be used for RNA ligation. Similar to T4 Rnl 1, T4 Rnl 2 also catalyzes the nucleophilic attack of the 3′-OH terminus onto the activated 5′-terminus to form a covalent 5′, 3′-phosphodiester bond. However, T4 Rnl 2 is much more active at joining nicks in double-stranded RNA (dsRNA) than at ligating the ends of ssRNA. Based on this feature, when the linear RNA precursor folds into a secondary structure with the ligation junction in a double-stranded region, the efficiency of T4 Rnl 2 is much higher than that of T4 Rnl 1. Besides, with the help of RNA splint, T4 Rnl 2 can also realize the ligation of ends of ssRNA.

Ribozyme Method

Nucleases are a class of RNAs with enzyme-catalyzed effects, and those applied to RNA cyclization include the PIE system (type I or type II introns) and other nucleases. The PIE system is one of the more commonly used methods, based on the self-scissoring function of type I or type II introns, which, in the presence of magnesium ions and free GTP, achieves a splicing effect that leads to the cyclization of the introns and the joining of intermediate sequences, resulting in the creation of a circRNA.

Fig. 4 Strategies for ribozyme methods. (Chen, X. et al., 2021)

• PIE system- I intron self-splicing

Group I intron self-splicing system requires only the addition of GTP and Mg2+ as cofactors and shows great potential for protein synthesis. This method realized RNA ligation through a normal group I intron self-splicing reaction, including two transesterifications at defined splice sites. The final circRNA will contain exogenous exon sequences.

• PIE system-II intron self-splicing

Group II intron self-splicing system involves the joining of the 5′ splice site at the end of an exon to the 3′ splice site at the beginning of the same exon. All exon sequences are dispensable for group II intron catalyzed inverse splicing. This method can enable more accurate linear RNA precursor ligation.

• Hairpin ribozymes

Hairpin ribozyme method can produce circRNA through the rolling circle reaction and the self-splicing reaction. The linear RNA precursor with HPR will fold into two alternative cleavage-active conformations to remove the 3′-end and the 5′-end. As a result, the intermediate will contain a 5′-OH and a 2′, 3′-cyclic phosphate to produce target circRNA.

Challenges and Outlooks

Although there are many ways to synthesize circRNAs in vitro, there are still many challenges and opportunities. CircRNA is still a hot area of research, and there are many difficulties to be solved.

Fig. 5 Challenges and potential solutions for circRNAs. (Chen, X. et al., 2021)

Faced with the growing demand for circRNAs, large quantities of circRNAs need to be produced. In a word, circRNA is still a hot area of current research and holds great potentials. With the continuous efforts in this field, circRNAs will play an essential role in basic research and medical applications, and become an important part of human health in the near future.