{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T11:47:02Z","timestamp":1782474422727,"version":"3.54.5"},"reference-count":12,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T00:00:00Z","timestamp":1781827200000},"content-version":"vor","delay-in-days":18,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T00:00:00Z","timestamp":1780272000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Self\u2010amplifying RNA (saRNA) derived from alphavirus replicons enables robust intracellular RNA amplification and high\u2010level protein expression at substantially lower doses than nonreplicating messenger RNA (mRNA) platforms. Venezuelan equine encephalitis virus (VEEV)\u2010based replicons lacking the viral structural genes are among the most extensively characterized saRNA backbones, combining efficient cytoplasmic replication with a favorable safety profile. However, incorporating chemical nucleoside modifications such as N1\u2010methylpseudouridine (m\n                    <jats:sup>1<\/jats:sup>\n                    \u03c8), now standard in conventional mRNA workflows, can directly impact replicase activity and hinder RNA amplification, necessitating backbone\u2010specific optimization. Recent studies have shown that incorporation of 5\u2010methylcytidine (m\n                    <jats:sup>5<\/jats:sup>\n                    C) preserves VEEV saRNA replication while reducing innate immune activation and improving expression durability. In an independent study, phosphatase treatment to remove residual 5\u2032\u2010triphosphates was shown to enhance saRNA functionality. Building on these findings, here we describe a streamlined protocol for generating m\n                    <jats:sup>5<\/jats:sup>\n                    C\u2010modified VEEV\u2010based saRNA using a single\u2010step in vitro transcription (IVT) strategy. This protocol utilizes PCR\u2010generated DNA templates with an encoded poly(A) tail, CleanCap AU for co\u2010transcriptional capping, and post\u2010transcriptional phosphatase treatment to minimize immunostimulatory RNA species. Finally, the RNA was purified using the phenol\u2010chloroform\u2010isoamyl alcohol method and functionally evaluated by transfection into HEK293T cells. This protocol provides a reproducible framework for producing capped, tailed, and chemically modified saRNA suitable for downstream functional and translational studies. \u00a9 2026 Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : IVT and purification of saRNA with modified nucleotides\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Functional assessment of IVT\u2013generated saRNA in HEK293T Cells\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70406","type":"journal-article","created":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T13:53:59Z","timestamp":1781877239000},"source":"Crossref","is-referenced-by-count":0,"title":["Streamlined In Vitro Transcription for Generating Self\u2010Amplifying RNA With Modified Nucleotides"],"prefix":"10.1002","volume":"6","author":[{"given":"Harish","family":"Prakash","sequence":"first","affiliation":[{"name":"Tata Institute for Genetics and Society  Bengaluru India"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karthik Kadhir","family":"Velu","sequence":"additional","affiliation":[{"name":"Tata Institute for Genetics and Society  Bengaluru India"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-5707-7917","authenticated-orcid":false,"given":"Vasanth","family":"Thamodaran","sequence":"additional","affiliation":[{"name":"Tata Institute for Genetics and Society  Bengaluru India"},{"name":"Academy of Scientific and Innovative Research  Ghaziabad India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,6,19]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41417\u2010022\u201000435\u20108"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1093\/narmme\/ugae004"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtn.2019.02.018"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2025.03.060"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1209367109"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpz1.39"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2005.06.008"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrd.2017.243"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2025.110487"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41587\u2010024\u201002306\u2010z"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mb0205as51"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.stem.2013.06.001"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70406","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.70406","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70406","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T11:04:21Z","timestamp":1782471861000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70406"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6]]},"references-count":12,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2026,6]]}},"alternative-id":["10.1002\/cpz1.70406"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70406","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6]]},"article-number":"e70406"}}