In Vitro Synthesis of circRNA

The application and development of circRNA in the therapeutic field benefit from continuous innovation in in vitro synthesis technologies and the constant optimization of methodologies. Currently, the in vitro synthesis of circRNA is primarily categorized into two major types: chemical synthesis and enzymatic synthesis. Enzymatic synthesis can be further subdivided into protein ligase-mediated synthesis, self-splicing ribozyme synthesis, and hairpin ribozyme-mediated synthesis.

Chemical Synthesis Method

The chemical synthesis of circRNA is mainly based on phosphoramidite chemistry and solid-phase synthesis methods, using nucleoside triphosphate derivatives as starting materials. This method enables the connection of 3',5'-phosphodiester bonds and effectively removes protective groups to prevent side reactions. However, current chemical synthesis technology can only produce circRNAs with a length of 70 to 80 nucleotides, which limits its potential application in the synthesis of long-fragment circRNAs.

Enzymatic Ligation Method

The enzymatic ligation method for synthesizing circRNA typically involves an in vitro transcription (IVT) reaction to prepare linear RNA precursors. This step requires a DNA template, an appropriate reaction buffer, and the collective action of phage RNA polymerases. Due to its relatively low cost and ability to synthesize longer RNA molecules, enzymatic ligation has become the mainstream in vitro RNA synthesis technology. After completing in vitro transcription, linear RNA precursors can be converted into circRNA using T4 RNA ligase (T4 Rnl) or ribozyme-catalyzed reactions.

(1) T4 Ligase Method

The T4 ligase family, including T4 DNA ligase (T4 Dnl), T4 Rnl 1, and T4 Rnl 2, plays a crucial role in catalyzing RNA ligation reactions by facilitating the connection between the 3'-OH acceptor region and the 5'-monophosphate donor region of linear RNA precursors. Therefore, it is necessary to form a 5'-monophosphate structure at the end of the precursor RNA during or after the IVT reaction. T4 Dnl is primarily used for the ligation of double-stranded DNA or DNA/RNA hybrids, while T4 Rnl 1 and T4 Rnl 2 are more suitable for joining single-stranded or double-stranded RNA.

To improve the efficiency of enzymatic ligation, splint sequences are often designed to pair with the 5' and 3' ends of the precursor RNA, or the ends themselves are designed into complementary structures. This brings the two ends into close proximity, thereby increasing the ligation efficiency of the T4 ligase.

(2) Ribozyme Method

The preparation of circRNA via the ribozyme method primarily relies on the self-splicing reactions of Group I and Group II introns, or the spontaneous ligation reaction of 5'-OH and 2',3'-cyclic phosphate intermediates formed by hairpin ribozyme (HPR) cleavage. The ribozyme method involves only the nucleic acid molecules themselves without the need for additional protein catalysts, making it highly suitable for process scale-up.

The Group I intron self-splicing system is a commonly used technology for the in vitro synthesis of circRNA. The synthesis system modified based on Group I intron self-splicing characteristics is known as the PIE (Permuted Intron-Exon) method, which requires only guanosine triphosphate (GTP) and magnesium ions (Mg2+) to achieve RNA circularization. In the PIE method, Group I intron sequences derived from the Anabaena tRNA Leu gene or the T4 phage thymidylate synthase (TS) gene are typically used; both have been proven to efficiently promote RNA circularization. Compared to chemical synthesis and T4 ligase methods, the PIE method is better suited for the circularization of larger linear RNA precursors, and its reaction conditions and purification processes are relatively simple. Consequently, the PIE method has become the most widely researched and utilized RNA circularization technology. Limitations of the PIE method include: variations in circularization efficiency across different RNA sequences due to precursor length and complex secondary structures; and the introduction of exogenous exon sequences which may alter the secondary structure of the circRNA, potentially leading to adverse effects on its activity and immunogenicity.

Group II self-splicing introns, after design and modification, form circRNA through back-splicing, which connects the 5' splice site at the end of an exon to the 3' splice site upstream of that exon. Unlike Group I introns, back-splicing catalyzed by Group II introns does not require additional GTP assistance to achieve linear RNA circularization.

HPR can utilize single-stranded circular DNA as a template to synthesize circRNA through a rolling circle reaction and a self-cleavage mechanism. During this process, the linear RNA precursor carrying the HPR folds into two types of conformations with cleavage activity. It cleaves the precursor RNA to generate an RNA intermediate with a 5'-OH group and a 2',3'-cyclic phosphate, which then forms circRNA through a spontaneous ligation reaction. This method is suitable for the efficient preparation of shorter circRNA molecules. However, it also has certain limitations: the cleavage and ligation reactions catalyzed by HPR are in a state of dynamic equilibrium, which may result in insufficient stability of the generated circRNA. Additionally, because the resulting circRNA contains partial HPR sequences, it may adversely affect the function of the circRNA.

In summary, circRNA synthesis methods based on self-splicing ribozyme technology-whether using Group I or Group II intron systems-are widely applied in scientific research and industrial production due to their simple reaction conditions, lack of length restrictions, and high product stability.