Therapeutic application of circular RNA aptamers in a mouse model of psoriasis

Data availability

All data supporting the findings of this study are available in the paper, Source Data and at https://doi.org/10.17632/zfm9kmfghs.2 (ref. 77). All sequencing data reported in this paper have been deposited in the Gene Expression Omnibus (GEO). High-throughput datasets generated in this study are available at GSE248680 (ref. 78), including circSHAPE-MaP data (GSE248679) and mouse RNA-seq data (IMQ-treated Pkr−/− mouse data, GSE248678; mouse spleens delivered EPIC-LNPs data, GSE253346); published RNA-seq data of patients with psoriasis can be downloaded from GEO under accession number GSE121212 (ref. 79). Source data are provided with this paper.

Code availability

This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact on request.

References

Hause, A. M. et al. Safety monitoring of bivalent COVID-19 mRNA vaccine booster doses among persons aged>/=12 years—United States, August 31-October 23, 2022. MMWR Morb. Mortal Wkly Rep.71, 1401–1406 (2022).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Winkle, M., El-Daly, S. M., Fabbri, M. & Calin, G. A. Noncoding RNA therapeutics—challenges and potential solutions. Nat Rev. Drug Discov.20, 629–651 (2021).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Enuka, Y. et al. Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor. Nucleic Acids Res.44, 1370–1383 (2016).

Article 
CAS 
PubMed 

Google Scholar 

Zhang, Y. et al. The biogenesis of nascent circular RNAs. Cell Rep.15, 611–624 (2016).

Article 
CAS 
PubMed 

Google Scholar 

Fischer, J. W., Busa, V. F., Shao, Y. & Leung, A. K. L. Structure-mediated RNA decay by UPF1 and G3BP1. Mol. Cell.78, 70–84.e76 (2020).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Liu, C. X. et al. Structure and degradation of circular RNAs regulate PKR activation in innate immunity. Cell177, 865–880.e821 (2019).

Article 
CAS 
PubMed 

Google Scholar 

Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell.74, 508–520.e504 (2019).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Liu, C. X. et al. RNA circles with minimized immunogenicity as potent PKR inhibitors. Mol. Cell.82, 420–434.e426 (2022).

Article 
CAS 
PubMed 

Google Scholar 

Liu, C. X. & Chen, L. L. Circular RNAs: characterization, cellular roles, and applications. Cell185, 2390 (2022).

Article 
CAS 
PubMed 

Google Scholar 

Bou-Nader, C., Gordon, J. M., Henderson, F. E. & Zhang, J. The search for a PKR code-differential regulation of protein kinase R activity by diverse RNA and protein regulators. RNA25, 539–556 (2019).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Cao, S. S., Song, B. & Kaufman, R. J. PKR protects colonic epithelium against colitis through the unfolded protein response and prosurvival signaling. Inflamm. Bowel Dis.18, 1735–1742 (2012).

Article 
PubMed 

Google Scholar 

Grolleau, A., Kaplan, M. J., Hanash, S. M., Beretta, L. & Richardson, B. Impaired translational response and increased protein kinase PKR expression in T cells from lupus patients. J. Clin. Invest.106, 1561–1568 (2000).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Rath, E. et al. Induction of dsRNA-activated protein kinase links mitochondrial unfolded protein response to the pathogenesis of intestinal inflammation. Gut61, 1269–1278 (2012).

Article 
CAS 
PubMed 

Google Scholar 

Zheng, X. & Bevilacqua, P. C. Activation of the protein kinase PKR by short double-stranded RNAs with single-stranded tails. RNA10, 1934–1945 (2004).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Ingrand, S. et al. The oxindole/imidazole derivative C16 reduces in vivo brain PKR activation. FEBS Lett.581, 4473–4478 (2007).

Article 
CAS 
PubMed 

Google Scholar 

Mouton-Liger, F. et al. PKR downregulation prevents neurodegeneration and β-amyloid production in a thiamine-deficient model. Cell Death Dis.6, e1594 (2015).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Watanabe, T. et al. Therapeutic effects of the PKR inhibitor C16 suppressing tumor proliferation and angiogenesis in hepatocellular carcinoma in vitro and in vivo. Sci. Rep.10, 5133 (2020).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Griffiths, C. E. M., Armstrong, A. W., Gudjonsson, J. E., & Barker, J. Psoriasis. Lancet397, 1301–1315 (2021).

Article 
CAS 
PubMed 

Google Scholar 

Moldovan, L. I. et al. High-throughput RNA sequencing from paired lesional- and non-lesional skin reveals major alterations in the psoriasis circRNAome. BMC Med. Genomics12, 174 (2019).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Moldovan, L. I. et al. Characterization of circular RNA transcriptomes in psoriasis and atopic dermatitis reveals disease-specific expression profiles. Exp. Dermatol.30, 1187–1196 (2021).

Article 
CAS 
PubMed 

Google Scholar 

Seeler, S. et al. Global circRNA expression changes predate clinical and histological improvements of psoriasis patients upon secukinumab treatment. PLoS ONE17, e0275219 (2022).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Swindell, W. R. et al. Imiquimod has strain-dependent effects in mice and does not uniquely model human psoriasis. Genome Med.9, 24 (2017).

Article 
PubMed 
PubMed Central 

Google Scholar 

Obi, P. & Chen, Y. G. The design and synthesis of circular RNAs. Methods196, 85–103 (2021).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Puttaraju, M. & Been, M. D. Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons. Nucleic Acids Res.20, 5357–5364 (1992).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun.9, 2629 (2018).

Article 
PubMed 
PubMed Central 

Google Scholar 

Guo, S. K., Nan, F., Liu, C. X., Yang, L. & Chen, L. L. Mapping circular RNA structures in living cells by SHAPE-MaP. Methods196, 47–55 (2021).

Article 
CAS 
PubMed 

Google Scholar 

Kuhen, K. L. et al. Structural organization of the human gene (PKR) encoding an interferon-inducible RNA-dependent protein kinase (PKR) and differences from its mouse homolog. Genomics36, 197–201 (1996).

Article 
CAS 
PubMed 

Google Scholar 

Samuel, C. E. The eIF-2 alpha protein kinases, regulators of translation in eukaryotes from yeasts to humans. J. Biol. Chem.268, 7603–7606 (1993).

Article 
CAS 
PubMed 

Google Scholar 

Wu, M. et al. lncRNA SLERT controls phase separation of FC/DFCs to facilitate Pol I transcription. Science373, 547–555 (2021).

Article 
CAS 
PubMed 

Google Scholar 

Yang, X. W. et al. MutS functions as a clamp loader by positioning MutL on the DNA during mismatch repair. Nat. Commun.13, 5808 (2022).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Hummert, J. et al. Photobleaching step analysis for robust determination of protein complex stoichiometries. Mol. Biol. Cell.32, ar35 (2021).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Yuan, J., He, K., Cheng, M., Yu, J. & Fang, X. Analysis of the steps in single-molecule photobleaching traces by using the hidden Markov model and maximum-likelihood clustering. Chem. Asian J.9, 2303–2308 (2014).

Article 
CAS 
PubMed 

Google Scholar 

Nallagatla, S. R., Toroney, R. & Bevilacqua, P. C. Regulation of innate immunity through RNA structure and the protein kinase PKR. Curr. Opin. Struct. Biol.21, 119–127 (2011).

Article 
CAS 
PubMed 

Google Scholar 

Heinicke, L. A., Nallagatla, S. R., Hull, C. M. & Bevilacqua, P. C. RNA helical imperfections regulate activation of the protein kinase PKR: effects of bulge position, size, and geometry. RNA17, 957–966 (2011).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Yan, Y., Tao, H., He, J. & Huang, S. Y. The HDOCK server for integrated protein-protein docking. Nat. Protoc.15, 1829–1852 (2020).

Article 
CAS 
PubMed 

Google Scholar 

Husain, B., Hesler, S. & Cole, J. L. Regulation of PKR by RNA: formation of active and inactive dimers. Biochemistry54, 6663–6672 (2015).

Article 
CAS 
PubMed 

Google Scholar 

Mayo, C. B. et al. Structural basis of protein kinase R autophosphorylation. Biochemistry58, 2967–2977 (2019).

Article 
CAS 
PubMed 

Google Scholar 

Dörner, T. & Furie, R. Novel paradigms in systemic lupus erythematosus. Lancet393, 2344–2358 (2019).

Article 
PubMed 

Google Scholar 

Tsokos, G. C., Lo, M. S., Costa Reis, P. & Sullivan, K. E. New insights into the immunopathogenesis of systemic lupus erythematosus. Nat. Rev. Rheumatol.12, 716–730 (2016).

Article 
CAS 
PubMed 

Google Scholar 

van der Fits, L. et al. Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. J. Immunol.182, 5836–5845 (2009).

Article 
PubMed 

Google Scholar 

Taylor, S. S., Haste, N. M. & Ghosh, G. PKR and eIF2alpha: integration of kinase dimerization, activation, and substrate docking. Cell122, 823–825 (2005).

Article 
CAS 
PubMed 

Google Scholar 

Ma, X. K. et al. CIRCexplorer3: a CLEAR pipeline for direct comparison of circular and linear RNA expression. Genom. Proteom. Bioinform.17, 511–521 (2019).

Article 

Google Scholar 

Futschik, M. E. & Carlisle, B. Noise-robust soft clustering of gene expression time-course data. J. Bioinform. Comput. Biol.3, 965–988 (2005).

Article 
CAS 
PubMed 

Google Scholar 

Kumar, L. & M, E. F. Mfuzz: a software package for soft clustering of microarray data. Bioinformation2, 5–7 (2007).

Article 
PubMed 
PubMed Central 

Google Scholar 

Lande, R. & Gilliet, M. Plasmacytoid dendritic cells: key players in the initiation and regulation of immune responses. Ann. N. Y. Acad. Sci.1183, 89–103 (2010).

Article 
CAS 
PubMed 

Google Scholar 

Chiricozzi, A., Romanelli, P., Volpe, E., Borsellino, G. & Romanelli, M. Scanning the immunopathogenesis of psoriasis. Int. J. Mol. Sci.19, 179 (2018).

Article 
PubMed 
PubMed Central 

Google Scholar 

Yang, Y. L. et al. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J.14, 6095–6106 (1995).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Nestle, F. O. et al. Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production. J. Exp. Med.202, 135–143 (2005).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Rácz, E. et al. Narrowband ultraviolet B inhibits innate cytosolic double-stranded RNA receptors in psoriatic skin and keratinocytes. Br. J. Dermatol.164, 838–847 (2011).

Article 
PubMed 

Google Scholar 

Zhang, L. J. et al. Antimicrobial peptide LL37 and MAVS signaling drive interferon-β production by epidermal keratinocytes during skin injury. Immunity45, 119–130 (2016).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Chen, L. L. et al. A guide to naming eukaryotic circular RNAs. Nat. Cell Biol.25, 1–5 (2023).

Article 
PubMed 
PubMed Central 

Google Scholar 

Zhang, X. O. et al. Complementary sequence-mediated exon circularization. Cell159, 134–147 (2014).

Article 
CAS 
PubMed 

Google Scholar 

Naidu, G. S. et al. A combinatorial library of lipid nanoparticles for cell type-specific mRNA delivery. Adv. Sci.10, e2301929 (2023).

Article 

Google Scholar 

Jones, S. A. et al. GILZ regulates Th17 responses and restrains IL-17-mediated skin inflammation. J. Autoimmun.61, 73–80 (2015).

Article 
CAS 
PubMed 

Google Scholar 

Fredriksson, T. & Pettersson, U. Severe psoriasis-oral therapy with a new retinoid. Dermatologica157, 238–244 (1978).

Article 
CAS 
PubMed 

Google Scholar 

Langley, R. G. & Ellis, C. N. Evaluating psoriasis with psoriasis area and severity index, psoriasis global assessment, and lattice system physician’s global assessment. J. Am. Acad. Dermatol.51, 563–569 (2004).

Article 
PubMed 

Google Scholar 

Chen, Y. G. et al. N6-methyladenosine modification controls circular RNA immunity. Mol. Cell.76, 96–109.e109 (2019).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Qu, L. et al. Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell185, 1728–1744.e1716 (2022).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Gal-Ben-Ari, S., Barrera, I., Ehrlich, M. & Rosenblum, K. PKR: a kinase to remember. Front. Mol. Neurosci.11, 480 (2018).

Article 
CAS 
PubMed 

Google Scholar 

Tronel, C., Page, G., Bodard, S., Chalon, S. & Antier, D. The specific PKR inhibitor C16 prevents apoptosis and IL-1beta production in an acute excitotoxic rat model with a neuroinflammatory component. Neurochem. Int.64, 73–83 (2014).

Article 
CAS 
PubMed 

Google Scholar 

Chang, R. C. et al. Involvement of double-stranded RNA-dependent protein kinase and phosphorylation of eukaryotic initiation factor-2alpha in neuronal degeneration. J. Neurochem.83, 1215–1225 (2002).

Article 
CAS 
PubMed 

Google Scholar 

Stern, E., Chinnakkaruppan, A., David, O., Sonenberg, N. & Rosenblum, K. Blocking the eIF2alpha kinase (PKR) enhances positive and negative forms of cortex-dependent taste memory. J. Neurosci.33, 2517–2525 (2013).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Zhu, P. J. et al. Suppression of PKR promotes network excitability and enhanced cognition by interferon-gamma-mediated disinhibition. Cell147, 1384–1396 (2011).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Feng, X. et al. Circular RNA aptamers ameliorate AD-relevant phenotypes by targeting PKR. Preprint at bioRxiv https://doi.org/10.1101/2024.03.27.583257 (2024).

Kaushik, S. B. & Lebwohl, M. G. Psoriasis: which therapy for which patient: psoriasis comorbidities and preferred systemic agents. J. Am. Acad. Dermatol.80, 27–40 (2019).

Article 
PubMed 

Google Scholar 

Manfreda, V., Esposito, M., Campione, E., Bianchi, L. & Giunta, A. Apremilast efficacy and safety in a psoriatic arthritis patient affected by HIV and HBV virus infections. Postgrad. Med.131, 239–240 (2019).

Article 
PubMed 

Google Scholar 

Guimaraes, C. P. et al. Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat. Protoc.8, 1787–1799 (2013).

Article 
PubMed 
PubMed Central 

Google Scholar 

Donovan, J., Rath, S., Kolet-Mandrikov, D. & Korennykh, A. Rapid RNase L-driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery. RNA23, 1660–1671 (2017).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Xiao, M. S. & Wilusz, J. E. An improved method for circular RNA purification using RNase R that efficiently removes linear RNAs containing G-quadruplexes or structured 3′ ends. Nucleic Acids Res.47, 8755–8769 (2019).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Matsui, T., Tanihara, K. & Date, T. Expression of unphosphorylated form of human double-stranded RNA-activated protein kinase in Escherichia coli. Biochem. Biophys. Res. Commun.284, 798–807 (2001).

Article 
CAS 
PubMed 

Google Scholar 

Smola, M. J., Rice, G. M., Busan, S., Siegfried, N. A. & Weeks, K. M. Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat. Protoc.10, 1643–1669 (2015).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Senavirathne, G. et al. Widespread nuclease contamination in commonly used oxygen-scavenging systems. Nat. Methods12, 901–902 (2015).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics30, 2114–2120 (2014).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods12, 357–360 (2015).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Kim, D. & Salzberg, S. L. TopHat-Fusion: an algorithm for discovery of novel fusion transcripts. Genome Biol.12, R72 (2011).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 

Zhang, Y., Wang, J. & Xiao, Y. 3dRNA: 3D structure prediction from linear to circular RNAs. J. Mol. Biol.434, 167452 (2022).

Article 
CAS 
PubMed 

Google Scholar 

Othniel, J. “doi: 10.17632/j6fmfjrc5y.1”, Mendeley Data, V1. Mendely Data https://doi.org/10.17632/94jg7jkt6n.1 (2020).

Guo, S. K. et al. Therapeutic Application of Circular RNA Aptamers in a Mouse Model of Psoriasis (Gene Expression Omnibus, 2024); http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE248680

Weidinger S., Rodriguez E., Tsoi L. C. & Gudjonsson J. Atopic Dermatitis, Psoriasis and Healthy Control RNA-seq Cohort (Gene Expression Omnibus, 2019); https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE121212

Download references

Acknowledgements

We thank F. Nan at Shanghai Institute of Materia Medica for support in LNPs, and the Chen laboratory members for critical discussion. This work was supported by National Key R&D Program of China (grant no. 2021YFA1300501), Strategic Priority Research Program of the Chinese Academy of Science (grant no. XDB0570000) and Science and Technology Commission of Shanghai Municipality (STCSM) (grant nos. 23DX1900100 and 23DX1900101) to L.-L.C.; National Natural Science Foundation of China (NSFC) (grant no. 31925011), National Key R&D Program of China (grant nos. 2021YFA1300503 and 2019YFA0802804) and STCSM (grant nos. 23DX1900102 and 23JS1400300) to L.Y.; NSFC (grant no. 32371349) and Shanghai Rising-Star Program (grant no. 23QA1410200) to C.-X.L.; China National Postdoctoral Program for Innovative Talents (grant nos. BX20220298 and BX20220077) and Shanghai Postdoctoral Excellence Program (grant nos. 2022757 and 2022728) to X.W. and F.N. D.P. acknowledges the support from the European Research Council (advance grant no. 101055029). This work has been supported by the New Cornerstone Science Foundation through the New Cornerstone Investigator Program and the XPLORER PRIZE.

Author information

Author notes

These authors contributed equally: Si-Kun Guo, Chu-Xiao Liu, Yi-Feng Xu, Xiao Wang, Fang Nan.

Authors and Affiliations

Key Laboratory of RNA Innovation, Science and Engineering, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China

Si-Kun Guo, Chu-Xiao Liu, Yi-Feng Xu, Xiao Wang, Youkui Huang, Siqi Li, Shan Nan, Ling Li, Chen Li, Meng-Yuan Wei, Jiaquan Liu & Ling-Ling Chen

Center for Molecular Medicine, Children’s Hospital of Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai, China

Fang Nan, Jia Wei & Li Yang

School of Life Science and Technology, ShanghaiTech University, Shanghai, China

Ling Li & Ling-Ling Chen

Laboratory of Precision Nanomedicine, Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Department of Materials Sciences and Engineering, Iby and Aladar Fleischman Faculty of Engineering, Center for Nanoscience and Nanotechnology, Cancer Biology Research Center, Tel Aviv University, Tel Aviv, Israel

Edo Kon, Nitay Ad-El & Dan Peer

Department of Dermatology, Beijing Chao-yang Hospital, Capital Medical University, Beijing, China

Rina Su & Shiguang Peng

National Institute of Biological Sciences, Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China

Ting Chen

New Cornerstone Science Laboratory, Shenzhen, China

Ling-Ling Chen

Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

Ling-Ling Chen

Contributions

L.-L.C. supervised and conceived the project. S.-K.G., C.-X.L., Y.-F.X. and X.W. designed and performed experiments. F.N. preformed computational analyses, supervised by L.Y. Y.H., S.L., L.L., E.K. and N.A. formulated LNPs, supervised by D.P. and L.-L.C. R.S. and S.P. provided samples from patients with psoriasis, supervised by T.C. C.L. helped with smTIRF experiments, supervised by J.L. J.W. generated the next-generation sequencing library. S.N. and M.-Y.W. helped with biochemical and mice experiments. L.-L.C., S.-K.G., C.-X.L., Y.-F.X., X.W. and F.N. wrote the paper with input from all authors. All authors read and approved the manuscript.

Corresponding author

Correspondence to
Ling-Ling Chen.

Ethics declarations

Competing interests

L.-L.C., S.-K.G., C.-X.L., S.L. and Y.-F.X. are named as inventors on patents related to circRNA held by CAS CEMCS. L.-L.C. is a scientific co-founder of RiboX Therapeutics. D.P. receives licensing fees (to patents on which he was an inventor) from, invested in, consults (or on scientific advisory boards or boards of directors) for, lectured (and received a fee) or conducts sponsored research at TAU for the following entities: ART Biosciences, BioNtech SE, Earli Inc., Kernal Biologics, Geneditor Biologics, Newphase Ltd, NeoVac Ltd, RiboX Therapeutics, Roche, SirTLabs Corporation and Teva Pharmaceuticals Inc.

Peer review

Peer review information

Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 EPIC synthesized by optimized strategy preserves its characteristics in minimized immunogenicity and folding status.

(a) Examination of circularization efficiency of two circular RNAs (circPOLR2A, 336 nt; circmCherry, 1452 nt) using Anabaena tRNALeu-derived PIE with varied lengths of extraneous nucleotides. Circularized RNA products are analyzed with denaturing PAGE, bands of RNA circles are verified with RNase R and marked with blue arrows. Representative results are shown from three replicates. Gels of each replicate are processed in parallel. (b) The 27 nt extraneous sequence tend to form stem-loop alone at the JS, independent of cargo sequences by predictions of in silico and SHAPE-MaP. (c) Ana_PIE_27nt version results in minimal induction of inflammatory factors. The same amounts of circular POLR2A (200 ng for each sample) with varied lengths of extraneous nucleotides were transfected into A549 cells. Relative expression of inflammatory factors after 6 hours transfection were examined by RT-qPCR. n = 3 biological repeats. (d) Secondary structures of circPOLR2A_Lig and circPOLR2A_J (EPIC). Left, the original and new junction sites are indicated in the secondary structure of circPOLR2A_Lig8. Right, the secondary structure of circPOLR2A_J (EPIC), in which the 27 nt extraneous sequences forms a stable step-loop. The predicted imperfect duplex regions are marked with gray and yellow shadows. (e) Human PKR protein purified from E. coli is shown by SDS-PAGE and Coomassie Blue staining. (f) The 27 extra nucleotides RNA circle doesn’t suppress PKR phosphorylation. Purified PKR (0.6 μM) is activated by 79 bp dsRNA (0.01 μM) in vitro, which is shown by autoradiography using γ-32P-ATP. 0.01 μM of RNA circles are used in the assays. (g) EPIC suppresses mouse PKR phosphorylation efficiently. Left, purified mouse PKR protein is shown by SDS-PAGE and Coomassie Blue staining. Right, the activation of mPkr (0.6 μM) by 79 bp dsRNA (0.01 μM) in vitro, is inhibited by EPIC (0.01 μM). c, f and g: n.s., p > 0.05, *p 
>>> Read full article>>>
Copyright for syndicated content belongs to the linked Source : Nature.com – https://www.nature.com/articles/s41587-024-02204-4