Precise Engineered circRNAs via Enzymatic Ligation Method
Publication Title: Generation of precise and accurate engineered circRNAs using enzymatic ligation
Summary
- Question
- This study aimed to develop a precise and efficient method for synthesizing engineered circular RNAs (circRNAs), which are RNA molecules with a closed-loop structure. CircRNAs have emerged as promising candidates for therapeutic applications due to their stability compared to linear RNAs, and they are increasingly used in biological research to mimic naturally occurring circRNAs. The researchers sought to address challenges related to unwanted extraneous sequences and inconsistencies in circRNA production.
- Why it Matters
- CircRNAs hold significant potential for advancing RNA-based therapeutics, including treatments for infectious diseases, neurological conditions, and cancer. Unlike linear RNAs, circRNAs resist degradation by exonucleases, enzymes that break down RNA, making them more stable and effective for clinical use. They can also drive efficient protein production and serve as tools for studying RNA biology. Precise circRNA synthesis is critical to avoid unintended sequences that can affect their biological function and therapeutic application.
- Methods
- The researchers employed an enzymatic ligation method to produce circRNAs, avoiding the use of autocatalytic splicing techniques that often introduce unwanted sequences. They optimized DNA templates to start transcription internally, reducing extraneous nucleotides. Linear RNA precursors were generated with accurate 5’ and 3’ ends, followed by enzymatic ligation using RNA ligase 2 and purification via urea–polyacrylamide gel electrophoresis (PAGE) to ensure high fidelity and eliminate contaminants.
- Key Findings
- The optimized workflow significantly improved the precision and yield of circRNA synthesis. RNA ligase 2 demonstrated the highest circularization efficiency, particularly when paired with modified DNA templates and RNA splints. The researchers achieved homogeneous circRNA preparations with accurate sequences and minimized contaminants. The method proved versatile, successfully applied to various RNA constructs of different lengths and sequences.
- Implications
- This precise circRNA synthesis method offers a reliable platform for advancing RNA-based therapeutics and biological research. By eliminating extraneous sequences and improving structural accuracy, the approach enhances the potential for circRNAs to be used in protein production, disease modeling, and therapeutic applications. The findings may help overcome barriers to widespread adoption of circRNA technologies in clinical and experimental settings.
- Next Steps
The researchers suggested further studies to compare the stability, translational efficiency, and immunogenicity of circRNAs synthesized using different methods. They also proposed expanding the application of their approach to explore its adaptability to diverse RNA sequences and therapeutic contexts.
- Funding Information
- This research was supported by the Rudolph J. Anderson Endowed Postdoctoral Fellowship, the National Institutes of Health (award R35GM142687). Additional support was provided by the Rita Allen Foundation and the Paul G. Allen Frontiers Group grant. Yale University also provided funding and support for this research.
Full Citation
Singh A, Dujsikova A, Mueller N, Chen Y. Generation of precise and accurate engineered circRNAs using enzymatic ligation. Nucleic Acids Research 2026, 54: gkag405. PMID: 42100852, PMCID: PMC13153714, DOI: 10.1093/nar/gkag405.
This AI-assisted summary has been reviewed and approved by at least one of the study's authors to ensure it accurately reflects the research.
Authors
Amrita Singh
First AuthorGrace Chen, PhD
Last AuthorAssistant Professor
Other Authors
Research Themes
Keywords
Concepts
- Enzymatic ligation;
- Urea-polyacrylamide gel electrophoresis;
- Autocatalytic splicing;
- Precursor RNA;
- Extraneous nucleotides;
- Reverse primers;
- RNA-based therapeutics;
- Linear RNAs;
- Transcription template;
- Gel electrophoresis;
- Degradation in vivo;
- Functional studies;
- RNA;
- Biological outcomes;
- Circular RNAs;
- CircRNAs;
- Sequence;
- Homogeneous sequences;
- RNA therapy;
- Internal guanosines;
- Nucleotide;
- Splicing;
- Primers;
- Transcription;
- Ribozyme