Methods for Preparation of Circular RNA

What is Circular RNA?

The COVID-19 pandemic has accelerated the development of mRNA vaccines, and the expedited approval of multiple vaccines has propelled the innovation of mRNA nucleic acid drugs. In 1976, Sanger and colleagues first identified a class of single-stranded covalently closed circular RNA molecules in plant viruses. Since 2012, with the advancement of high-throughput sequencing technologies, a large number of functional circular RNAs have continuously emerged over the past decade. Covalently closed circular structures can protect circular RNA from degradation by nucleases, rendering circular RNA more structurally stable compared to mRNA. Circular RNA translates proteins via a cap-independent mechanism, serving as mRNA 2.0 and providing possibilities for developing vaccines that are more efficient than mRNA-based ones.

General structure of circular RNA.

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What are Applications of Circular RNA in Vaccine Development?

Circular RNA shares advantages over mRNA vaccines compared to traditional vaccines. It enables standardized production and the development of new products through sequence optimization, significantly reducing production costs. Circular RNA also possesses a more stable structure than mRNA, making it resistant to RNAase degradation and greatly reducing storage and transportation costs. Due to its natural stable structure, circular RNA can translate proteins at high levels without the need for nucleotide modifications, thus avoiding the influence of innate immunity in the body.

What are Methods for Preparation of Circular RNA?

The method of artificial synthesis of circular RNA is crucial for harnessing the potential advantages of circular RNA technology over mRNA and realizing its promising industrial prospects. Currently, there are three commonly used methods for the artificial synthesis of circular RNA, including self-splicing of Group I introns (from T4 bacteriophage or Anabaena), self-splicing of Group II introns, and the T4 RNA ligase method.

Group I Intron (from T4 Bacteriophage or Anabaena) Self-Splicing

Previously, the method of Group I intron self-splicing has been widely used for in vitro circularization of short RNA sequences, with circularization efficiencies exceeding 80% for sequences ranging from 58 to 124 nucleotides. However, for longer sequences (over 1500 nucleotides), the circularization efficiency cannot meet the requirements for mass production. One approach involves using two types of introns for self-splicing to artificially prepare circular RNA, incorporating exonic sequences from phage and cyanobacteria, each with lengths of 74 nt and 186 nt, respectively. By designing a self-splicing intron and incorporating auxiliary sequences that facilitate splicing, it becomes possible to efficiently circularize RNA sequences exceeding 1500 nt in length in vitro. Additionally, through sequence optimization, circular RNA preparation utilizing T4 bacteriophage can be achieved without leaving residual exogenous sequences. However, this method has low efficiency and currently cannot meet industrial demands.

Group II Intron Self-Splicing

Using the self-catalyzed splicing reaction of Group II introns from yeast, circular RNA can be artificially synthesized in vitro. By reversing the sequences of regions D1-D3 and D5-D6 within the six structural domains of Group II introns, self-splicing ribozymes can be formed, facilitating the production of circular RNA in vitro. Circular RNAs prepared using this method do not retain residual exogenous sequences. However, the efficiency of this method is low, and industrial-scale production remains challenging at present. The self-splicing reaction of Group II introns from Clostridium tetani bacteria can also be used to artificially synthesize circular RNA in vitro. The natural self-splicing of Clostridium tetani Group II introns may introduce additional sequences from the PIE system into the target molecule. Through sequence design, the self-splicing system of Clostridium tetani Group II introns can achieve circular RNA preparation without leaving residual exogenous sequences, similar to the yeast Group II intron splicing system. However, the efficiency of circular RNA preparation without residual exogenous sequences still needs to be improved.

Sequence Redesign for in Vitro Circular RNA Synthesis

Traditional methods using T4 DNA or T4 RNA ligase with the aid of splint sequences can achieve in vitro circular RNA preparation. However, these methods suffer from low efficiency and challenging purification processes, making industrial-scale production difficult. By computationally simulating RNA structures and identifying the optimal sites for RNA circularization, it is possible to achieve circularization of RNA without the need for additional splint or auxiliary strands. This method significantly suppresses the production of large molecular byproducts during the cyclization process, with preparation efficiency reaching up to 93%. This approach enables efficient circularization of RNA sequences that were previously difficult or unable to be circularized. The concept of sequence redesign at the circularization interface is not only applicable to T4 ligase-based methods but also compatible with efficient in vitro circular RNA preparation using intron self-splicing systems.

Tool Enzymes in Circular RNA Preparation

The preparation process for in vitro circular RNA has matured, and it primarily involves the use of key enzymes during large-scale production. These enzymes include T7 RNA polymerase, T4 RNA ligase 1/2, ssRNA ligase, circGFP, circLuc standards, etc. The flexible use of enzymes greatly facilitates the production process, accelerating the application and industrialization of circular RNA. Simultaneously, the elimination of residual linear RNA interference is crucial for the artificial preparation of high-purity circular RNA. Circular RNA is resistant to digestion by RNase R, and efficient RNase R can completely remove linear RNA. RNase R is not only a key ingredient in the circular RNA artificial preparation using the ligation method but also indispensable in the intron splicing-based circular RNA artificial preparation methods.

T7 Thermoscript RNA Polymerase

T7 Thermoscript RNA Polymerase can carry out in vitro transcription at higher temperatures. It can efficiently transcribe at temperatures ranging from 37 to 52°C, with the optimal temperature being 37°C. Even under conditions of 50°C, it retains over 60% of its activity, while the wild-type enzyme is inactive at this temperature. In a standard 20 µL reaction system, using 1 µg of the Control template provided with the reagent as a template can yield 150 to 200 µg of product, with transcription lengths of up to 5000 nt achievable.

T4 RNA Ligase 1

T4 RNA Ligase 1, also known as T4 Rnl 1, is an ATP-dependent enzyme that catalyzes the formation of phosphodiester bonds between the 5'-P end and the 3'-OH end of single-stranded RNA, single-stranded DNA, or nucleotide molecules, both intermolecularly and intramolecularly.

T4 RNA Ligase 2

T4 RNA Ligase 2, also known as T4 Rnl 2, is an ATP-dependent double-stranded RNA ligase that can be used for both intramolecular circularization and intermolecular linear ligation of double-stranded RNA molecules. Unlike T4 RNA Ligase 1, T4 RNA Ligase 2 exhibits significantly higher activity in connecting nicks within double-stranded RNA than in connecting the ends of single-stranded RNA. T4 RNA Ligase 2 can also be used for the connection of the 3' hydroxyl group of RNA chains and the 5' phosphate group of DNA chains within or between double-stranded nucleic acid molecules (double-stranded RNA, RNA/DNA hybrids, or double-stranded DNA). T4 RNA Ligase 2 requires the presence of a 5' phosphate and a 3' hydroxyl group for ligation, enabling the connection reaction to occur between the 5' phosphate group of either an RNA or DNA chain and the 3' hydroxyl group of an RNA chain.

ssRNA Cyclase

ssRNA Cyclase is a thermostable ligase that catalyzes the ligation (i.e., cyclization) of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA) substrates that possess both a 5'-monophosphate and a 3'-hydroxyl group. Linear ssDNAs and ssRNAs greater than 15 nucleotides in length can be cyclized by ssRNA Cyclase. In the cyclization reaction, it is required that the catalytic substrates, single-stranded DNA/RNA, have a 5'-phosphate group and a 3'-OH group. This enzyme efficiently connects substrates that are greater than 15 nucleotides in length.

RNase R

RNase R (Ribonuclease R) is a 3'-5' exoribonuclease derived from the RNR superfamily of Escherichia coli. It can sequentially cleave RNA from the 3'-5' direction into dinucleotides and trinucleotides. RNase R can digest nearly all linear RNA molecules but is less effective at digesting circular RNA, hairpin structures, or double-stranded RNA molecules with less than 7 nucleotides of 3' overhangs.