Breaking the Production Bottleneck of circRNA: Byproduct Analysis and Efficient Cyclization Strategies

Breaking the Production Bottleneck of circRNA: Byproduct Analysis and Efficient Cyclization Strategies

CircRNA, a star of the RNA 2.0 era, stands out due to its unique covalently closed-loop structure. Compared to traditional mRNA, circRNA offers higher stability, longer expression duration, and lower required dosage, which has garnered significant attention. However, its production process still contains many steps that need optimization, particularly with regard to byproducts formed during the process. Many circRNAs synthesized in labs show strong immune responses, low expression levels, and short half-lives in cellular experiments, with these issues often stemming from byproduct formation during synthesis.

CircRNA Byproducts 

During the synthesis of circRNA, various byproducts can be formed depending on the reaction conditions, which significantly impact the functionality and efficiency of the circRNA. Below are the main byproduct types and their characteristics:

• Linear Precursor RNA (Precursor RNA): Linear precursor RNA refers to RNA molecules that fail to fully cyclize during circRNA synthesis. Its length varies depending on the cyclization strategy: in T4 RNA ligase-based cyclization reactions, the unreacted linear precursor RNA is of the same length as the circRNA; while in ribozyme-based PIE or STS cyclization reactions, the linear precursor RNA is typically longer than the circRNA.
• Nicked circRNA: When the circular structure of a circRNA is disrupted, nicked linear or partially linear RNA fragments are formed. These fragments may be the same length as the original circRNA, or they may be shorter due to multiple breaks.
• Introns: In ribozyme-mediated PIE or STS cyclization reactions, intron sequences excised during circRNA synthesis may not be fully removed. Some intron fragments may carry or lack 3' phosphate groups at their 5' ends.
• High Molecular Weight RNA (HMW): Byproducts consisting of double-stranded RNA (dsRNA) or other RNA aggregates.
• circRNA Aggregates: These aggregates consist of multiple circRNA molecules clustering together, potentially affecting the spatial conformation of circRNA and its proper distribution within cells.

Purification Analysis of circRNA and Its Byproducts

Impact of CircRNA Byproducts on Functionality 

Byproducts formed during circRNA production have a significant impact on its stability, expression efficiency, and functional application. Below are the specific effects of major byproducts on circRNA functionality:

• Impact on Expression Efficiency and Immune Response: Linear precursor RNA, nicked circRNA, and high molecular weight RNA significantly interfere with circRNA expression and immune responses:

1. Impact on Expression Efficiency: Linear precursor RNA competes for transcription resources with circRNA during synthesis. An excess of linear precursor RNA significantly reduces circRNA synthesis efficiency, lowering its expression level in cells, thereby limiting its functional potential. Moreover, the unique circular structure of circRNA is crucial for its stability and function. Nicked circRNA loses this structural feature, exposing linear ends that make it more susceptible to degradation by nucleases, leading to a drastically shortened half-life and affecting its sustained activity within cells.
2. Immune Response: Exogenous, unmodified circRNA, due to its unique circular structure, can efficiently evade cellular RNA sensors, thus avoiding immune activation in cells or mice with active RIG-I and Toll-like receptors (TLRs). However, high molecular weight RNA and small amounts of linear precursor RNA with 3' phosphate groups are recognized by intracellular pattern recognition receptors, triggering immune signaling pathways and provoking a strong immune response. This response not only interferes with circRNA’s normal function but may also significantly reduce its effectiveness in practical applications.

• Impact on Processing and Transport: Residual introns negatively affect the processing and transport of circRNA. During circRNA synthesis, introns must be completely excised to ensure proper processing and transport. If introns are not fully removed, this can hinder circRNA’s movement and localization within the cell, preventing it from reaching its functional sites and directly impairing its utility. Additionally, some intron fragments may possess regulatory functions, interacting with other molecules and indirectly affecting circRNA function. This interference may become more apparent in specific contexts.
• Impact on Functional Activity: The formation of circRNA aggregates has a significant impact on its function. The presence of these aggregates can alter the spatial conformation of circRNA, making it difficult to properly bind to target molecules and hindering functional activity. Furthermore, aggregates can affect the distribution of circRNA within cells, preventing it from being correctly localized to its intended action sites. This abnormal distribution not only diminishes the efficacy of circRNA but may also induce off-target effects, leading to potential risks.

Optimization Strategies for CircRNA Production Processes

Levostar is committed to in-depth research and systematic optimization of the circRNA production process. Through scientific process improvements, we have significantly reduced the generation of common byproducts and greatly enhanced the expression efficiency of circRNA. These optimizations not only improve product quality but also further promote the application of circRNA in research and drug development.

1. Reaction Condition Optimization: Taking the PIE system with intron type I as an example, Levostar has conducted comprehensive exploration and optimization of key reaction conditions, significantly improving cyclization efficiency:

• Magnesium ions and free GTP content adjustment: Optimizing the ratio of key cofactors in the IVT (in vitro transcription) system ensures a more efficient cyclization reaction and reduces the amount of linear precursor RNA.
• Fine control of reaction time and temperature: Systematic experiments identify the best reaction time and temperature combinations to prevent RNA degradation or byproduct formation caused by excessively long reaction times.
• Optimization of linearized plasmid template amount: Precisely controlling the starting amount of the linearized template reduces the negative impact of unreacted templates on cyclization efficiency.

Before optimization of the reaction system

After optimization of the reaction system

2. Downstream Purification Process Improvements

Levostar has developed a unique and efficient purification strategy that significantly enhances the stability and expression efficiency of circRNA while drastically reducing the likelihood of immune reactions. For example, by transfecting circRNA-eGFP into H293T cells for testing, we verified the effect of the optimized production process. Experimental results show that circRNA expression levels in cells were significantly higher than those produced using conventional methods. Furthermore, the circRNA, after optimization of both the IVT system and downstream purification process, exhibited expression levels comparable to traditional mRNA, proving the excellent performance of this optimized process in improving circRNA expression.

Comparison of eGFP Expression across Different Processes

 (A: circRNA before optimization; B: circRNA after IVT system optimization; C: circRNA after IVT system and purification process optimization; D: eGFP-mRNA)

Through continuous technical exploration and innovation, Levostar has made breakthrough progress in optimizing circRNA production processes. Our self-developed reaction condition optimization strategy significantly enhances cyclization efficiency, while our unique purification process greatly improves product stability and expression efficiency, effectively reducing the risk of immune responses.