RESUMO
We show that oligodeoxyribonucleotides (oligos) composed of alpha- and beta-anomeric sections can be used as antisense compounds. An octamer has been chosen as an effector domain to form a substrate for RNaseH. This octamer is complementary to the translation start site of the pim-1 protooncogene mRNA. Chimeric alpha-beta oligos and their beta-analogs have a similar binding affinity for their target. These oligos also direct efficient RNaseH-mediated cleavage of target mRNA. Among all alpha-beta oligos studied, one with an alpha-fragment bound by its 3'-end to the 3'-end of the beta-octamer is the most resistant to nucleolytic digestion in biological media. The alpha-beta oligos have been found to inhibit in vitro translation of pim-1 RNA with specificity.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Animais , Sequência de Bases , Sangue , Meios de Cultura , Eritroblastos , Globinas/genética , Humanos , Hidrólise , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Ribonuclease H/metabolismoRESUMO
A new type of chimeric oligonucleotides composed of alpha- and beta-sections is described. The sequence of eight beta-nucleotides flanked at 3'- or/and 5'-ends by nuclease-resistant alpha-oligonucleotides has been chosen as an effector domain to form a substrate for RNase H. The synthesized oligonucleotides are complementary to the translation initiation site of the pim protooncogene mRNA. We used the chemical ligation method to prepare the chimeric oligonucleotides. The thermal stability of heteroduplexes formed by the alpha beta oligonucleotides with a complementary strand is not significantly altered compared to that of their beta-analogs. These oligonucleotides promote efficient RNase H-mediated cleavage of pim mRNA. Among the alpha beta oligonucleotides studied, one with an alpha-fragment bound by its 3'-end to the 3'-end of the beta-octanucleotide proved to be the most resistant to nucleolytic digestion in human plasma, calf serum, and murine fibroblast lysate. This alpha beta oligonucleotide directs more specific RNA cleavage by RNase H than its beta beta counterpart.