RESUMO
The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.
Assuntos
Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/química , Células 3T3 , Animais , Eletroforese em Gel de Poliacrilamida , Endonucleases/metabolismo , Exonucleases/metabolismo , Vírus da Leucemia Murina de Friend/genética , Genes env/genética , Células HeLa , Humanos , Camundongos , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligorribonucleotídeos/química , Ribonuclease H/metabolismo , Espectrofotometria Ultravioleta , Termodinâmica , TransfecçãoRESUMO
We studied the uptake and intracellular distribution of an FITC labelled phosphodiester oligodeoxynucleotide (ODN) vectorized by a dendrimeric structure in cell culture.
Assuntos
Oligonucleotídeos/metabolismo , Poliaminas/química , Células 3T3 , Animais , Fluoresceína-5-Isotiocianato , Células HeLa , Humanos , Camundongos , Microscopia ConfocalRESUMO
Factors limiting the use of antisense phosphodiester oligodeoxynucleotides (ODNs) as therapeutic agents are inefficient cellular uptake and intracellular transport to RNA target. To overcome these obstacles, ODN carriers have been developed, but the intracellular fate of ODNs is controversial and strongly depends on the means of vectorization. Polyamidoamine dendrimers are non-linear polycationic cascade polymers that are able to bind ODNs electrostatically. These complexes have been demonstrated to protect phosphodiester ODNs from nuclease degradation and also to increase their cellular uptake and pharmacological effectiveness. We studied the intracellular distribution of a fluorescein isothiocyanate-labeled ODN vectorized by a dendrimer vector and found that intracellular ODN distribution was dependent on the phase of the cell cycle, with a nuclear localization predominantly in the G2/M phase. In addition, in order to evaluate the relevance of ODN vectors in enhancing the inhibition of the targeted genes' expression, we developed a rapid screening system which measures the transient expression of two reporter genes, one used as target, the other as control and vice versa. This system was validated through investigating the effect of the dendrimer vector on ODN biological activity. Antisense sequence-specific inhibition of more than 70% of one reporter gene was obtained with a chimeric ODN containing four phosphorothioate groups, two at each end.
