RESUMEN
Complementary oligodeoxynucleotides (ODNs) that contain 2-aminoadenine and 2-thiothymine interact weakly with each other but form stable hybrids with unmodified complements. These selectively binding complementary (SBC) agents can invade duplex DNA and hybridize to each strand (Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., and Gamper, H. B. (1996) Biochemistry 35, 11170-11176). Antisense ODNs with similar properties should be less encumbered by RNA secondary structure. Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H. SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs. Optimal binding was observed when the entire hairpin sequence was targeted. Translation of L. amazonensis mRNA in a cell-free extract was more efficiently inhibited by SBC ODNs complementary to the mini-exon hairpin than by the corresponding NB ODNs. Nonspecific protein binding in the cell-free extract by phosphorothioate SBC ODNs rendered them ineffective as antisense agents in vitro. SBC phosphorothioate ODNs displayed a modest but significant improvement of leishmanicidal properties compared with NB phosphorothioate ODNs.
Asunto(s)
Exones , Leishmania/genética , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , Animales , Secuencia de Bases , Mapeo Cromosómico , Calor , Datos de Secuencia Molecular , Tionucleótidos/metabolismoRESUMEN
G/A motif triplex-forming oligonucleotides (TFOs) complementary to a 21 base pair homopurine/homopyrimidine run were conjugated at one or both ends to chlorambucil. These TFOs were incubated with several synthetic duplexes containing the targeted homopurine run flanked by different sequences. The extent of mono and interstrand cross-linking was compared with the level of binding at equilibrium. Covalent modification took place within a triple-stranded complex and usually occurred at guanine residues in the flanking double-stranded DNA. The efficiency of alkylation was dependent upon the sequence of the flanking duplex, the solution conditions, and the rate of triplex formation relative to the rate of chlorambucil reaction. Self-association of the TFOs as parallel duplexes was demonstrated and this did not interfere with triple strand formation. With an optimal target, cross-linking of the triplex was very efficient when incubation was carried in a physiological buffer supplemented with the triplex selective intercalator coralyne.