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

Kellan T. Passow, Nicole M. Antczak, Shana J. Sturla, Daniel A. Harki

Research output: Contribution to journalArticlepeer-review

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 languageEnglish (US)
Article numbere101
JournalCurrent protocols in nucleic acid chemistry
Volume80
Issue number1
DOIs
StatePublished - Mar 1 2020

Bibliographical note

Funding 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)

Publisher Copyright:
© 2020 John Wiley & Sons, Inc.

Keywords

  • 4-cyanoindole
  • fluorescent nucleosides
  • polymerase incorporation

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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