Custom Circular RNA (circRNA) Synthesis Service

Introduction

Linear RNA constructs face challenges in terms of stability and expression levels in preclinical and clinical studies. Our Custom Circular RNAs Synthesis Service addresses these via advanced enzymatic and ribozyme-based circularization, providing highly stable, long-lasting, efficiently translated circular RNAs. Custom-designed, high-purity, and application-optimized, it supports vaccine development, protein expression, and gene therapy, aiding progress from concept to clinic.

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Custom Circular RNAs Synthesis Service

Once considered non-functional byproducts of gene transcription, circular RNAs (circRNAs) are now recognized as a widespread and functionally significant class of RNA molecules. Generated through a process called back-splicing, these covalently closed RNA loops are exceptionally stable, lacking the free 5' and 3' ends that are typically targeted by exonucleases. Their unique structure allows them to persist in cells far longer than their linear counterparts, making them ideal candidates for a wide range of therapeutic applications, from protein expression to vaccine development.

Design of messenger circRNA vectors

Fig.1 Rational design of circRNA sequences.¹

• Translation initiation element selection and optimization: Prioritize elements derived from CVB3, echovirus 29, human rhinovirus B, or enterovirus B for strong activity in pancreatic cancer cells. Enhance efficiency by inserting eIF4G-associated aptamers.
• Spacer design: to reduce structural hindrance, ensuring unimpeded ribosome recruitment.
• Minimizing RNA duplexes: Design sequences to avoid duplex formation, preventing PKR-mediated degradation, and maintaining circRNA stability.
• UTR optimization: Include UTRs with PABP binding sites, poly (A)/poly (AC) sequences, PABP motifs, and eIF4G-recruiting aptamers to boost translation and reduce immunogenicity.
• Codon optimization: Match codons to pancreatic cancer cell tRNA abundance; eliminate adverse base-pairing between the translation initiation element and coding sequences for efficient translation and proper folding.
• RBPs motifs introduction: Incorporate translation-enhancing RBPs motifs to elevate protein expression in pancreatic cancer cells.

Synthesis of Exogenous Circular RNAs (circRNAs)

Exogenous circRNA synthesis primarily involves two approaches: in vivo transcription and in vitro circularization, with specific strategies and characteristics as follows:

• In VivoTranscription

Fig.2 Types of endogenous and synthetic circRNA backbones.²

• Utilize introns from endogenously highly expressed circRNAs to mediate circularization via back-splicing. However, this may generate a mixture of circRNAs and linear RNAs, requiring construct optimization to enhance specificity.
• Introduce cis-acting elements or trans-acting factors to mimic natural splicing mechanisms, promoting splice site proximity and improving circularization efficiency.
• Construct vectors using viral promoters (e.g., CMV) and inverted repeats, or combine ribozymes for circularization. Note that some methods may leave residual "scar sequences."

• In Vitro Circularization
  • Intron-based systems: Such as the permuted intron-exon (PIE) system, which employs group I self-splicing introns for circularization. Applicable both in vivo and in vitro, but may retain exogenous sequences.
  • Scarless strategies: Avoid non-target sequence residues and reduce immunogenicity via rearranging exon sequences of T4 td introns, optimizing the D1 domain of group II introns, or utilizing the trans-splicing activity of Tetrahymena group I introns.
  • Chemical/enzyme catalysis: Linear precursors are circularized via T4 DNA ligase, T4 RNA ligases, or chemical reagents. Suitable for short sequences but with low yield.

Workflow

1. Required Starting Materials:
   1. The target RNA sequence or a plasmid containing the desired gene sequence.
   2. Details of the intended application (vaccine development, protein expression, etc.).
   3. Any specific design constraints or optimization preferences (e.g., specific regulatory elements or modified nucleotides).
2. Sequence Optimization & Plasmid Design: Expert analysis and optimization of sequences for stability and translation efficiency; design of optimal in vitro transcription plasmids.
3. In Vitro Transcription: Linear RNA transcription from designed plasmids using a robust T7 system.
4. Circularization: Efficient circularization via enzymatic ligation and ribozyme-mediated self-splicing, with a focus on scarless strategies for purity.
5. Purification: Meticulous purification (e.g., HPLC) to remove linear RNA, enzymes, and impurities.
6. Quality Control: Rigorous testing: gel electrophoresis, RNase R resistance, UV spectrophotometry, and sequencing to confirm identity and integrity.
7. Final Deliverables: Upon completion, you will receive:
   1. Purified, high-quality circular RNA molecules.
   2. A detailed Certificate of Analysis (CoA) outlining purity, concentration, and quality control results.
   3. A comprehensive Project Report summarizing the synthesis process, optimization strategies used, and a summary of all QC data.
8. Estimated Timeframe: The typical timeframe for our synthesis service ranges from 6 to 10 weeks, depending on the complexity of the sequence and the level of optimization required. Rush services are available upon request.

What We Can Offer

Creative Biolabs is a leader in the synthesis of custom circular RNAs, providing a seamless, end-to-end service designed to meet the rigorous demands of modern biological research and therapeutic development.

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Case Study

Circularity of the RNAs	Microscopic imaging of the translation product
Fig.3 RNA cyclization was verified using denatured PAGE.2	Fig.4 The translation of circular RNA within cells was observed using an inverted fluorescence microscope.2

FAQs

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References

1. Choi, Seo-Won, and Jin-Wu Nam. "Optimal design of synthetic circular RNAs." Experimental & Molecular Medicine 56.6 (2024): 1281-1292. https://doi.org/10.1038/s12276-024-01251-w. Distributed under Open Access license CC BY 4.0, without modification.
2. Abe, Naoko, et al. "Rolling circle translation of circular RNA in living human cells." Scientific Reports 5.1 (2015): 16435. https://doi.org/10.1038/srep16435. Distributed under Open Access license CC BY 4.0, the pictures were cropped.