Kellan T. Passow, Nicole M. Antczak, Shana J. Sturla, Daniel A. Harki
下载PDF
{"title":"Synthesis of 4-Cyanoindole Nucleosides, 4-Cyanoindole-2ʹ-Deoxyribonucleoside-5ʹ-Triphosphate (4CIN-TP), and Enzymatic Incorporation of 4CIN-TP into DNA","authors":"Kellan T. Passow, Nicole M. Antczak, Shana J. Sturla, Daniel A. Harki","doi":"10.1002/cpnc.101","DOIUrl":null,"url":null,"abstract":"<p>4-Cyanoindole-2ʹ-deoxyribonucleoside (4CIN) is a fluorescent isomorphic nucleoside analogue with superior spectroscopic properties in terms of Stokes shift and quantum yield in comparison to the widely utilized isomorphic nucleoside analogue, 2-aminopurine-2ʹ-deoxyribonucleoside (2APN). Notably, when inserted into single- or double-stranded DNA, 4CIN experiences substantially less in-strand fluorescence quenching compared to 2APN. Given the utility of these properties for a spectrum of research applications involving oligonucleotides and oligonucleotide-protein interactions (e.g., enzymatic processes, DNA hybridization, DNA damage), we envision that additional reagents based on 4-cyanoindole nucleosides may be widely utilized. This protocol expands on the previously published synthesis of 4CIN to include synthetic routes to both 4-cyanoindole-ribonucleoside (4CINr) and 4-cyanoindole-2ʹ-deoxyribonucleoside-5ʹ-triphosphate (4CIN-TP), as well as a method for the enzymatic incorporation of 4CIN-TP into DNA by a polymerase. These methods are anticipated to further enable the utilization of 4CIN in diverse applications involving DNA and RNA oligonucleotides. © 2020 by John Wiley & Sons, Inc.</p><p><b>Basic Protocol 1</b>: Synthesis of 4-cyanoindole-2ʹ-deoxyribonucleoside (4CIN) and 4CIN phosphoramidite <b>4</b></p><p><b>Basic Protocol 2</b>: Synthesis of 4-cyanoindole-ribonucleoside (4CINr)</p><p><b>Basic Protocol 3</b>: Synthesis of 4-cyanoindole-2ʹ-deoxyribonucleoside-5ʹ-triphosphate (4CIN-TP)</p><p><b>Basic Protocol 4</b>: Steady state incorporation kinetics of 2AP-TP and 4CIN-TP by a DNA polymerase</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2020-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.101","citationCount":"4","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Nucleic Acid Chemistry","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpnc.101","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Chemistry","Score":null,"Total":0}
引用次数: 4
引用
批量引用
Abstract
4-Cyanoindole-2ʹ-deoxyribonucleoside (4CIN) is a fluorescent isomorphic nucleoside analogue with superior spectroscopic properties in terms of Stokes shift and quantum yield in comparison to the widely utilized isomorphic nucleoside analogue, 2-aminopurine-2ʹ-deoxyribonucleoside (2APN). Notably, when inserted into single- or double-stranded DNA, 4CIN experiences substantially less in-strand fluorescence quenching compared to 2APN. Given the utility of these properties for a spectrum of research applications involving oligonucleotides and oligonucleotide-protein interactions (e.g., enzymatic processes, DNA hybridization, DNA damage), we envision that additional reagents based on 4-cyanoindole nucleosides may be widely utilized. This protocol expands on the previously published synthesis of 4CIN to include synthetic routes to both 4-cyanoindole-ribonucleoside (4CINr) and 4-cyanoindole-2ʹ-deoxyribonucleoside-5ʹ-triphosphate (4CIN-TP), as well as a method for the enzymatic incorporation of 4CIN-TP into DNA by a polymerase. These methods are anticipated to further enable the utilization of 4CIN in diverse applications involving DNA and RNA oligonucleotides. © 2020 by John Wiley & Sons, Inc.
Basic Protocol 1 : Synthesis of 4-cyanoindole-2ʹ-deoxyribonucleoside (4CIN) and 4CIN phosphoramidite 4
Basic Protocol 2 : Synthesis of 4-cyanoindole-ribonucleoside (4CINr)
Basic Protocol 3 : Synthesis of 4-cyanoindole-2ʹ-deoxyribonucleoside-5ʹ-triphosphate (4CIN-TP)
Basic Protocol 4 : Steady state incorporation kinetics of 2AP-TP and 4CIN-TP by a DNA polymerase
4-氰吲哚核苷、4-氰吲哚-2′-脱氧核糖核苷-5′-三磷酸(4CIN-TP)的合成及4CIN-TP在DNA中的酶促结合
4-氰吲哚-2′-脱氧核糖核苷(4CIN)是一种荧光同形核苷类似物,与广泛使用的同形核苷类似物2-氨基嘌呤-2′-脱氧核糖核苷(2APN)相比,在斯托克斯位移和量子产率方面具有优越的光谱特性。值得注意的是,当插入单链或双链DNA时,与2APN相比,4CIN的链内荧光猝灭明显更少。鉴于这些特性在涉及寡核苷酸和寡核苷酸-蛋白质相互作用的一系列研究应用中的效用(例如,酶促过程,DNA杂交,DNA损伤),我们设想基于4-氰吲哚核苷的其他试剂可能被广泛使用。该方案扩展了先前发表的4CIN合成,包括合成4-氰吲哚-核糖核苷(4CINr)和4-氰吲哚-2′-脱氧核糖核苷-5′-三磷酸(4CIN- tp)的途径,以及通过聚合酶将4CIN- tp酶结合到DNA中的方法。这些方法有望进一步使4CIN在涉及DNA和RNA寡核苷酸的各种应用中得到利用。©2020 by John Wiley &基本方案1:合成4-氰吲哚-2 α -脱氧核糖核苷(4CIN)和4CIN磷酰胺。基本方案2:合成4-氰吲哚-2 α -脱氧核糖核苷(4CINr)。基本方案3:合成4-氰吲哚-2 α -脱氧核糖核苷-5 α -三磷酸(4CIN- tp)。基本方案4:2AP-TP和4CIN- tp通过DNA聚合酶的稳态掺入动力学
本文章由计算机程序翻译,如有差异,请以英文原文为准。