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.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health (NIH; R01‐GM110129 to DAH), the Swiss National Science Foundation (310030_185020), and a 3M Graduate Research Fellowship to KTP. Mass spectrometry was performed at the Analytical Biochemistry Core Facility of the University of Minnesota Masonic Cancer Center, which is supported by the NIH (P30‐CA77598)
© 2020 John Wiley & Sons, Inc.
- fluorescent nucleosides
- polymerase incorporation
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't