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

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.