Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

Kira Astakhova, Santhosh T. Kumar, Jesper Wengel

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Herein, a novel fluorescent nucleotide analogue, perylene-2'-amino-alpha-L-LNA, has been prepared and studied within synthetic oligonucleotides of different sequences. The phosphoramidite reagent was synthesized in 85% overall yield starting from 2'-amino-alpha-L-LNA nucleoside. Incorporation efficiency of the resulting perylene-2'-amino-alpha-L-LNA monomer (T*) into synthetic oligonucleotides was significantly improved by replacement of the typically used 1H-tetrazole activator with pyridine hydrochloride. Generally, oligonucleotides containing monomer T* showed high binding affinity towards complementary DNA and RNA targets, batochromically shifted excitation/emission wavelengths with respect to the often applied polyaromatic hydrocarbon pyrene, high fluorescent quantum yields and very low target detection limits (5-10 nM). Fluorescence of single stranded LNA/DNA mixmer oligonucleotide having two incorporations of monomers T* was quenched (quantum yield Phi(F) = 0.21) relative to duplexes of this probe with complementary DNA and RNA (Phi(F) = 0.42 and 0.35, respectively). On the contrary, a strong fluorescence quenching upon target binding was demonstrated by two short oligonucleotides of analogues sequences containing monomers T* at 5'- and 3'-terminal positions. We explain the hybridization-induced light-up effect observed for double-labeled probe by a reduction of fluorescence quenching due to precise positioning of the fluorophores within the double-stranded complexes. Furthermore, we propose that a covalent link between two T* monomers in the double-labeled probe provides a remarkable degree of rigidity in the double helix which enforces positioning of the bulky perylene moieties in the nonpolar groove resulting in reduced fluorescence quenching.
Original languageEnglish
JournalCollection of Czechoslovak Chemical Communications
Volume76
Issue number11
Pages (from-to)1347-1360
Number of pages14
ISSN0010-0765
DOIs
Publication statusPublished - 2011
Externally publishedYes

Keywords

  • DNA
  • Fluorescent probes
  • Oligonucleotides
  • Nucleosides
  • Nucleotides
  • LNA
  • Molecular diagnostics

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