Purification of circular RNA
RNA Circularization: Circular RNAs (circRNAs) are single-stranded RNAs with a contiguous structure that have enhanced stability and lack end motifs necessary for interaction with various cellular proteins. CircRNAs are important thereapeutic target, as they are less susceptible to degradation by cellular exonucleases, which typically target the ends of linear RNA molecules. As a result, circRNAs can persist longer in cells than their linear counterparts.
Additionally, unmodified exogenous circRNA has the ability to bypass cellular RNA sensors and avoid provoking an immune response. With it's extended translation duration, circRNAs make promising candidates for use in protein production applications and have significant implications for the development of new therapies and treatments.
The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity.
circRNA Purification: Purifying circular RNAs (circRNAs) can be challenging. The purification process involves several steps, including RNase R digestion, high-performance liquid chromatography (HPLC), and phosphatase treatment. Even small amounts of contaminating linear RNA can lead to robust cellular immune responses, so it is important to ensure that the purification process is thorough and effective. However, with careful attention to detail and the use of appropriate techniques, it is possible to obtain highly purified circRNA preparations.
Sepax Technologies, Inc. large pore SRT size exclusion chromatography (SEC) is an excellent choice for analytical and prep purification to remove non-circular components from circRNA samples.
Analytical (CV of 4.99 mL)
Company Name:? MIT School of Science , Juvenile Diabetes Research Foundation (JDRF) , MIT Koch Institute for Integrative Cancer Research , The National Institutes of Health
Authors:? R. Alexander Wesselhoeft , Piotr Kowalski , Daniel G. Anderson , Frances C Parker-Hale, Yuxuan Huang , Namita Bisaria, PhD, MBA
Year:?2019
Separation Mode:?SEC
Product:?HPLC Column, Sepax, SRT SEC-2000 PEEK, 5um, 2000 A 4.6 x 300 mm
Part Number:?215980P-4630
Specific Sample:?circRNA
Molecular Weight of Sample:?1-6kb
Mobile Phase:?RNA was run in RNase-free TE buffer (10mM Tris, 1mM EDTA, pH:6)
Flow Rate:?0.3mL/minute
Instrument:?HPLC
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Instrument Notes:?Agilent 1100 Series, RNA was detected by UV absorbance at 260nm, but was collected without UV detection
Full Title:?RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo
Citation:?Wesselhoeft, R. Alexander, et al. "RNA circularization diminishes immunogenicity and can extend translation duration in vivo." Molecular cell 74.3 (2019): 508-520.
Link to Paper:?https://www.sciencedirect.com/science/article/pii/S10
Preparative (CV of 212.06 mL)
Company Name:?Suzhou CureMed Biopharma Technology
Authors: Jiali?Yang,?Jiafeng?Zhu,?Yiyun?Chen,?Yaran?Du,?Yiling?Tan,?Linpeng?Wu,?Jiaojiao?Sun,?Mengting?Zhai,?Lixiang?Wei,?Na?Li,?Ke?Huang,?Qiangbo?Hou,?Zhenbo?Tong,?Andreas?Bechthold,?Zhenhua?Sun, Chijian Zuo
Year:?2021
Separation Mode:?SEC
Product:?HPLC Column, Sepax, SRT SEC-1000, 5um, 1000 A 30 x 300 mm
Part Number:?215950-30030
Specific Sample:?circRNA
Mobile Phase:?RNase-free Phosphate buffer (pH 6)
Flow Rate:?15mL/min
Full Title:?Intratumoral Delivered Novel Circular mRNA Encoding Cytokines for Immune Modulation and Cancer Therapy
Citation:?Yang, Jiali, et al. "Intratumoral Delivered Novel Circular mRNA Encoding Cytokines for Immune Modulation and Cancer Therapy." bioRxiv (2021).
Link to Paper:?https://doi.org/10.1101/2021.11.01.466725