{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T09:16:01Z","timestamp":1788513361197,"version":"build-2803163510"},"reference-count":31,"publisher":"Oxford University Press (OUP)","issue":"16","license":[{"start":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T00:00:00Z","timestamp":1788480000000},"content-version":"vor","delay-in-days":11,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Medical Technology Development Program"},{"DOI":"10.13039\/501100001321","name":"National Research Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001321","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Korean government","award":["2022M3E5F1017657"],"award-info":[{"award-number":["2022M3E5F1017657"]}]},{"name":"Rznomics Inc"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,24]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Circular RNAs (circRNAs) are more stable than linear RNAs, enabling expanding applications in RNA vaccines and therapeutics. We previously developed an in vitro circRNA preparation method based on end-to-end self-targeting and splicing (STS) using the Tetrahymena group I intron, which generates circRNAs without extraneous sequences. However, self-circularization efficiency declines as gene of interest (GOI) length increases, limiting its application to longer GOIs. Here, we systematically optimized key determinants of STS efficiency, including target site selection and P1 construct engineering. Target site screening revealed that selection of optimal target sites within each GOI markedly improved self-circularization efficiency. Moreover, engineering of the P1 construct, including incorporation of a polyA10 sequence upstream of the internal guide sequence of the intron and an antisense sequence complementary to the target site and its upstream region at the 5\u2032 side of polyA10, further enhanced efficiency. Notably, the optimized STS strategy achieved up to two-fold higher self-circularization efficiency than the conventional permuted intron\u2013exon (PIE) method for long GOIs (\u223c8 K-nt). Collectively, these results establish an improved STS workflow for efficient circRNA production without extraneous sequences across a wide range of GOI lengths, outperforming the PIE method for long GOIs, and broadening biomedical applications.<\/jats:p>","DOI":"10.1093\/nar\/gkag869","type":"journal-article","created":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T08:41:21Z","timestamp":1788511281000},"source":"Crossref","is-referenced-by-count":0,"title":["Target site selection and P1 engineering enable highly efficient circular RNA production via end-to-end self-targeting and splicing"],"prefix":"10.1093","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8246-7592","authenticated-orcid":false,"given":"Kyung Hyun","family":"Lee","sequence":"first","affiliation":[{"name":"R&D Center, Rznomics Inc. , Seongnam 13468 ,","place":["Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seongcheol","family":"Kim","sequence":"additional","affiliation":[{"name":"R&D Center, Rznomics Inc. , Seongnam 13468 ,","place":["Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juwon","family":"Cha","sequence":"additional","affiliation":[{"name":"R&D Center, Rznomics Inc. , Seongnam 13468 ,","place":["Republic of Korea"]},{"name":"Department of Bioconvergence Engineering, Research Institute of Advanced Omics, Dankook University , Yongin 16890 ,","place":["Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1718-7601","authenticated-orcid":false,"given":"Seong-Wook","family":"Lee","sequence":"additional","affiliation":[{"name":"R&D Center, Rznomics Inc. , Seongnam 13468 ,","place":["Republic of Korea"]},{"name":"Department of Bioconvergence Engineering, Research Institute of Advanced Omics, Dankook University , Yongin 16890 ,","place":["Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,9,4]]},"reference":[{"key":"2026090404411565500_B1","doi-asserted-by":"publisher","first-page":"1281","DOI":"10.1038\/s12276-024-01251-w","article-title":"Optimal design of synthetic circular RNAs","volume":"56","author":"Choi","year":"2024","journal-title":"Exp Mol Med"},{"key":"2026090404411565500_B2","doi-asserted-by":"publisher","first-page":"1838","DOI":"10.1242\/dev.128074","article-title":"Circular RNAs: analysis, expression and potential functions","volume":"143","author":"Barrett","year":"2016","journal-title":"Development"},{"key":"2026090404411565500_B3","doi-asserted-by":"publisher","first-page":"e1478","DOI":"10.1002\/wrna.1478","article-title":"A 360 degree view of circular RNAs: from biogenesis to functions","volume":"9","author":"Wilusz","year":"2018","journal-title":"Wiley Interdiscip Rev RNA"},{"key":"2026090404411565500_B4","doi-asserted-by":"publisher","first-page":"2016","DOI":"10.1016\/j.cell.2022.04.021","article-title":"Circular RNAs: characterization, cellular roles, and applications","volume":"185","author":"Liu","year":"2022","journal-title":"Cell"},{"key":"2026090404411565500_B5","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1038\/s41576-024-00806-x","article-title":"The therapeutic potential of circular RNAs","volume":"23","author":"O\u2019Leary","year":"2025","journal-title":"Nat Rev Genet"},{"key":"2026090404411565500_B6","doi-asserted-by":"publisher","first-page":"1728","DOI":"10.1016\/j.cell.2022.03.044","article-title":"Circular RNA vaccines against SARS-CoV-2 and emerging variants","volume":"185","author":"Qu","year":"2022","journal-title":"Cell"},{"key":"2026090404411565500_B7","doi-asserted-by":"publisher","first-page":"e2302191120","DOI":"10.1073\/pnas.2302191120","article-title":"Circular RNA vaccine induces potent T cell responses","volume":"12","author":"Amaya","year":"2023","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026090404411565500_B8","doi-asserted-by":"publisher","first-page":"e2406434121","DOI":"10.1073\/pnas.2406434121","article-title":"CXCL13 promotes broad immune responses induced by circular RNA vaccines","volume":"121","author":"Wan","year":"2024","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026090404411565500_B9","doi-asserted-by":"publisher","first-page":"341","DOI":"10.1038\/s41392-023-01561-x","article-title":"Circular RNA vaccine in disease prevention and treatment","volume":"8","author":"Nie","year":"2023","journal-title":"Signal Transduct. 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