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Notably, when inserted into single\u2010 or double\u2010stranded DNA, 4CIN experiences substantially less in\u2010strand fluorescence quenching compared to 2APN. Given the utility of these properties for a spectrum of research applications involving oligonucleotides and oligonucleotide\u2010protein interactions (e.g., enzymatic processes, DNA hybridization, DNA damage), we envision that additional reagents based on 4\u2010cyanoindole nucleosides may be widely utilized. This protocol expands on the previously published synthesis of 4CIN to include synthetic routes to both 4\u2010cyanoindole\u2010ribonucleoside (4CINr) and 4\u2010cyanoindole\u20102\u02b9\u2010deoxyribonucleoside\u20105\u02b9\u2010triphosphate (4CIN\u2010TP), as well as a method for the enzymatic incorporation of 4CIN\u2010TP into DNA by a polymerase. These methods are anticipated to further enable the utilization of 4CIN in diverse applications involving DNA and RNA oligonucleotides. \u00a9 2020 by John Wiley &amp; Sons, Inc.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Synthesis of 4\u2010cyanoindole\u20102\u02b9\u2010deoxyribonucleoside (4CIN) and 4CIN phosphoramidite <jats:bold>4<\/jats:bold><\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Synthesis of 4\u2010cyanoindole\u2010ribonucleoside (4CINr)<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Synthesis of 4\u2010cyanoindole\u20102\u02b9\u2010deoxyribonucleoside\u20105\u02b9\u2010triphosphate (4CIN\u2010TP)<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Steady state incorporation kinetics of 2AP\u2010TP and 4CIN\u2010TP by a DNA polymerase<\/jats:p>","DOI":"10.1002\/cpnc.101","type":"journal-article","created":{"date-parts":[[2020,1,7]],"date-time":"2020-01-07T18:09:30Z","timestamp":1578420570000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Synthesis of 4\u2010Cyanoindole Nucleosides, 4\u2010Cyanoindole\u20102\u02b9\u2010Deoxyribonucleoside\u20105\u02b9\u2010Triphosphate (4CIN\u2010TP), and Enzymatic Incorporation of 4CIN\u2010TP into DNA"],"prefix":"10.1002","volume":"80","author":[{"given":"Kellan T.","family":"Passow","sequence":"first","affiliation":[{"name":"Department of Medicinal Chemistry University of Minnesota  Minneapolis Minnesota"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicole M.","family":"Antczak","sequence":"additional","affiliation":[{"name":"Department of Health Sciences and Technology ETH Zurich  Zurich Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shana J.","family":"Sturla","sequence":"additional","affiliation":[{"name":"Department of Health Sciences and Technology ETH Zurich  Zurich Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel A.","family":"Harki","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry University of Minnesota  Minneapolis Minnesota"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2020,1,7]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules24030602"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978-1-85617-567-8.50011-1"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00092a039"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/cr990045m"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1529\/biophysj.105.071837"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-9216-4_19"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/jhet.5570250202"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.200700160"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi026120w"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi7021848"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1039\/P19840000657"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.7b00154"},{"key":"e_1_2_7_14_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201712726"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.200600128"},{"key":"e_1_2_7_16_1","first-page":"131","article-title":"A new route to nucleoside 5\u02b9\u2010triphosphates","volume":"16","author":"Ludwig J.","year":"1981","journal-title":"Acta Biochimica Et Biophysica; 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