Synthesis of 4-Cyanoindole Nucleosides, 4-Cyanoindole-2ʹ-Deoxyribonucleoside-5ʹ-Triphosphate (4CIN-TP), and Enzymatic Incorporation of 4CIN-TP into DNA

Q4 Chemistry
Kellan T. Passow, Nicole M. Antczak, Shana J. Sturla, Daniel A. Harki
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引用次数: 4

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
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来源期刊
Current Protocols in Nucleic Acid Chemistry
Current Protocols in Nucleic Acid Chemistry Chemistry-Organic Chemistry
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期刊介绍: Published in association with International Society for Nucleosides, Nucleotides & Nucleic Acids (IS3NA) , Current Protocols in Nucleic Acid Chemistry is equally valuable for biotech, pharmaceutical, and academic labs. It is the resource for designing and running successful research projects in the rapidly growing and changing field of nucleic acid, nucleotide, and nucleoside research.
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