Real-time detection and efficacy of antisense oligonucleotides delivered by PAMAM dendrimers in living cells

Abstract

The aim of the present investigation was to study the behavior of PAMAM dendrimer-nucleic acid complexes in vitro and living cells. We demonstrated the rapid and sensitive detection of mRNA in living cells using molecular beacon pair, one with a donor and the other with a quenching fluorophore that hybridises to adjacent regions on the same mRNA target, resulting in fluorescence resonance energy transfer (FRET). The molecular beacon was composed of a 13-nt loop structure containing the antisense sequence that can hybridise with the AUG translational start site of the Friend env gene. It was shown that SuperFect may stimulate the antisense ON-RNA hybridisation. The secondary structure of antisense oligonucleotide was optimized. An antisense sequence-specific inhibition of 75% was obtained for one reporter gene with a stem-loop ODN containing four phosphorothioate groups, two at each end.