Interfacing Click Chemistry with Automated Oligonucleotide Synthesis for the Preparation of Fluorescent DNA Probes Containing Internal Xanthene and Cyanine Dyes

Kira Astakhova, Jesper Wengel

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2'-O-propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid-phase copper(I)-catalyzed azidealkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40110 nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to
Original languageEnglish
JournalChemistry - A European Journal
Volume19
Issue number3
Pages (from-to)1112-1122
Number of pages11
ISSN0947-6539
DOIs
Publication statusPublished - 2013
Externally publishedYes

Keywords

  • Bioanalysis
  • Click chemistry
  • Fluorescence
  • Nucleic acids
  • Sensors

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