SPECIFIC-INHIBITION OF NITRIC-OXIDE PRODUCTION IN MACROPHAGES BY PHOSPHOROTHIOATE ANTISENSE OLIGONUCLEOTIDES

The effects of antisense oligonucleotides (ODNs) on nitric oxide (NO) production induced by lipopolysaccharide (LPS) were investigated using thioglycollate-induced mouse peritoneal macrophages. Antisense phosph orothioate ODNs (S-oligo) corresponding to a sequence in the neighborh ood of the AUG initiation codon of a mouse inducible nitric oxide synt hase (iNOS) mRNA, which has a G-quartet motif in its antisense sequenc e, inhibited NO induction in a dose-dependent manner. Antisense phosph odiester ODNs (D-oligo), 5'- and 3'-terminal phosphorothioate-modified antisense ODNs and control scramble and missense S-oligos had no such effect. In addition, control nonsense and two mismatched S-oligos, wh ich include G-quartet motif in their sequences, inhibited NO induction to similar to 50% of those in the control. Antisense S-oligo showed t he inhibitory effect on NO production by exposure of macrophages to va rious concentrations of LPS. Western blot analysis using anti-mouse in ducible nitric oxide synthase (iNOS) antibody revealed that antisense S-oligo specifically removed an immunoreactive band at 130 kDa. In add ition, the results of reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effect originated from a specific reduction of the targeted iNOS mRNA by hybridization with the antisen se S-oligo. Furthermore, no ODNs affected beta-actin mRNA and tumor ne crosis factor alpha (TNF-alpha) expression in macrophages stimulated b y LPS. These findings demonstrated that antisense S-oligo inhibited NO production derived from iNOS expression in macrophages by an antisens e mechanism, including the aptameric effect partially mediated by the G-quartet motif.