Collect. Czech. Chem. Commun. 2011, 76, 1347-1360
Published online 2011-11-20 19:55:15

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

Irina V. Astakhovaa,*, T. Santhosh Kumara,b and Jesper Wengela

a Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark
b Present address: Chemical Biology Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8, Rm. 121, NIH, NIDDK, LCBB, Bethesda, MD 20892-0810, USA


Herein, a novel fluorescent nucleotide analogue, perylene-2′-amino-α-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-α-L-LNA nucleoside. Incorporation efficiency of the resulting perylene-2′-amino-α-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 ΦF = 0.21) relative to duplexes of this probe with complementary DNA and RNAF = 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.

Keywords: DNA; Fluorescence; Fluorescent probes; Oligonucleotides; Nucleosides; Nucleotides; LNA; Molecular diagnostics.

References: 45 live references.