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.
Original language | English (US) |
---|---|
Article number | e101 |
Journal | Current protocols in nucleic acid chemistry |
Volume | 80 |
Issue number | 1 |
DOIs | |
State | Published - Mar 1 2020 |
Bibliographical note
Publisher Copyright:© 2020 John Wiley & Sons, Inc.
Keywords
- 4-cyanoindole
- fluorescent nucleosides
- polymerase incorporation