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The synthesis of the cnm<jats:sup>5<\/jats:sup>U phosphoramidite building block starts with commercially available 5\u2010methyluridine (m<jats:sup>5<\/jats:sup>C), followed by bromination of the 5\u2010methyl group to install the cyano moiety using TMSCN\/TBAF. The cn<jats:sup>5<\/jats:sup>U phosphoramidite is obtained by regular Vorbr\u00fcggen glycosylation of the protected ribofuranose with silylated 5\u2010cyanouracil. These two modified phosphoramidites are suitable for synthesis of RNA oligonucleotides on solid phase using conventional amidite chemistry. Our protocol provides access to two novel building blocks for constructing RNA\u2010based therapeutics. \u00a9 2020 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Preparation of cnm<jats:sup>5<\/jats:sup>U and cn<jats:sup>5<\/jats:sup>U phosphoramidites<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Synthesis, purification, and characterization of cnm<jats:sup>5<\/jats:sup>U\u2010 and cn<jats:sup>5<\/jats:sup>U\u2010modified RNA oligonucleotides<\/jats:p>","DOI":"10.1002\/cpnc.114","type":"journal-article","created":{"date-parts":[[2020,8,27]],"date-time":"2020-08-27T05:35:30Z","timestamp":1598506530000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Synthesis of 5\u2010Cyanomethyluridine (cnm<sup>5<\/sup>U) and 5\u2010Cyanouridine (cn<sup>5<\/sup>U) Phosphoramidites and Their Incorporation into RNA Oligonucleotides"],"prefix":"10.1002","volume":"82","author":[{"given":"Song","family":"Mao","sequence":"first","affiliation":[{"name":"Department of Chemistry and The RNA Institute University at Albany State University of New York  Albany New York"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hsu\u2010Chun","family":"Tsai","sequence":"additional","affiliation":[{"name":"Department of Chemistry and The RNA Institute University at Albany State University of New York  Albany New York"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jia","family":"Sheng","sequence":"additional","affiliation":[{"name":"Department of Chemistry and The RNA Institute University at Albany State University of New York  Albany New York"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2020,8,26]]},"reference":[{"key":"e_1_2_5_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2006.11.046"},{"key":"e_1_2_5_3_1","doi-asserted-by":"publisher","DOI":"10.1039\/C6OB02674E"},{"key":"e_1_2_5_4_1","first-page":"165","volume-title":"tRNA: Structure, biosynthesis, and function, American Society for Microbiology","author":"Bj\u04e7rk G. 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