circRNA sponges are artificial, non-coding miRNA sponges derived from in vitro transcription (IVT) and circularization by permutated intron exon (PIE) or enzymatic ligation. Multiple miRNA recognizing elements (MREs) are integrated within the synthetic circRNA to increase miRNA sponging capability mimicking the endogenous circRNAs. Compared to linear RNAs, circRNA sponges lacks 5’ cap and 3’ poly(a) tail and are more resistant to exonucleolytic degradation resulting in higher intracellular levels for miRNA target derepression. circRNA sponges overcomes the toxicity exhibited by anti-miRNA oligonucleotides (AMO) and dosage constraint by the plasmid-based miRNA sponge. circRNA sponges does not require nucleoside modifications and are inherently non-immunogenic. However, in the context of PIE-derived circRNA, N6-methyladenosine (M6A) nucleoside modification can further minimize immune recognition. Moreover, size-exclusion HPLC, phosphatase dephosphorylation, and RNase R degradation can be used to remove production impurities which trigger innate immune response.
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| miR-122-5p circRNA sponge absorbs miR-122 and derepress TGFβR1 transcription in mouse cells | |
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| Western blot analysis of TGFβ receptor 1 (TGFβR1) in Hepa1-6 cell when treated artificial circRNA sponge for miR-122 (122sp) and untreated as an negative control (NC) . The silencing of miR-122 by circRNA sponge in Hepa1-6 increase TGFβR1 expression. | |
| miR-122-5p circRNA sponge affinity pulldown assay | |
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| E-Gel™ EX 2% Agarose Gel of circRNA pulldown. Lane 1: The binding complex, miR-122-5p circRNA sponge 313nt and biotinylated miR-122-5p, was pulled down by Streptavidin Magnetic Microspheres. Lane 2: miR-122-5p circRNA sponge alone not pulled down by Streptavidin Magnetic Microspheres. |
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