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

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