title: Design of potent phosphorothioate antisense oligonucleotides directed to human interleukin 10 gene product and their evaluation of antisense activity in U937 cells.
abstract: OBJECTIVE
  The two objectives of this study were to design potent phosphorothioate antisense oligonucleotides (AS-S-oligos) directed against the human interleukin-10 (IL-10) gene product and to reveal the DNA sequence which best activates antisense effects.
  
  
  METHODS
  The design of potent AS-S-oligo was performed by using melting temperature (Tm) value of a DNA/RNA hybrid calculated by the nearest neighbor method and a secondary structure of human IL-10 mRNA suggested by RNA folding algorithms. U937 cells were used to estimate the antisense effect of the AS-S-oligos.
  
  
  RESULTS
  Of the eight candidates selected as potent AS-S-oligos on the basis of having higher Tm values and favorable secondary structures of the IL-10 mRNA, AS-S-oligos directed against the translated (AS367-S-oligo) and 3'-untranslated (AS637-S-oligo) region of IL-10 mRNA showed the strongest inhibitory effects on IL-10 production and this inhibition was dose- and time-dependent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effects of AS-S-oligos originated from a specific reduction of target IL-10 mRNA by hybridization with AS367- and AS637-S-oligos. In addition, these AS-S-oligos did not affect human tumor necrosis factor-alpha (TNF-alpha) production in the cells stimulated by lipopolysaccharide (LPS). Strong positive correlations between the inhibitory effect of AS-S-oligos on the IL-10 production and not only Tm values calculated by nearest neighbor method but also Tm values determined by absorbance versus temperature profiles were demonstrated except for AS25-S-oligo and AS1249-S-oligo.
  
  
  CONCLUSIONS
  These findings suggest AS367- and AS637-S-oligos powerfully inhibit IL-10 production in U937 cells via an antisense mechanism. In addition, it is suggested efficiency of AS-S-oligo directed against the sequence of the target gene product can be explained by these Tm values and the proposed secondary structures of the target gene product.
authors.affiliation: School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Arima
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
authors.forename: Y
authors.initials: Y
authors.lastname: Aramaki
authors.forename: T
authors.initials: T
authors.lastname: Sakamoto
authors.forename: K
authors.initials: K
authors.lastname: Yuda
authors.forename: K
authors.initials: K
authors.lastname: Akiyama
authors.forename: T
authors.initials: T
authors.lastname: Goto
authors.forename: S
authors.initials: S
authors.lastname: Tsuchiya
mesh_terms: D015195:Drug Design; D005069:Evaluation Studies as Topic; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D013873:Thionucleotides / Q000138:chemical synthesis / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects / Q000378:metabolism; D020298:U937 Cells
chemical_list: D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013873:Thionucleotides; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10