The reduction of Raf-1 protein by phosphorothioate ODNs and siRNAs targeted to the same two mRNA sequences.
Two sets of 20-mer phosphorothioate-modified oligodeoxynucleotide DNAs (sODN) and 21-mer or 22-mer small interfering RNAs (siRNAs), targeted to the same coding sites in raf-1 mRNA, were compared for their abilities to reduce the amount of endogenously expressed Raf-1 protein in T24 cells. The amount of Raf-1 protein was monitored by careful quantitation of Western blots. We found that the siRNAs were somewhat less effective than the S-ODNs in reducing the Raf-1 protein level 20 hours after a 4-hour transfection. The siRNA duplexes were characterized by circular dichroism (CD) spectra, and melting temperatures (Tm) were obtained for the siRNA duplexes and DNA x RNA hybrids formed by the S-ODNs. The S-ODNs differed in their effectiveness, the S-ODN that formed the more stable hybrid being the more effective in reducing the Raf-1 protein level, but the two siRNAs were equally effective despite a difference in Tm of about 20 degrees C. Finally, the siRNAs and S-ODNs had a comparable nonspecific effect on a nontargeted (Bcl-2) protein. Our data add to others in the literature that show it can be difficult to select siRNAs that are more effective than antisense ODNs in downregulating endogenously expressed proteins.
Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083-0688, USA.
Tzu-Fang
TF
Lou
Carla W
CW
Gray
Donald M
DM
Gray
D001483:Base Sequence; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000494:pharmacology*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D013816:Thermodynamics; D013873:Thionucleotides / Q000494:pharmacology*; D014162:Transfection; D001749:Urinary Bladder Neoplasms
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013873:Thionucleotides; D019908:Proto-Oncogene Proteins c-raf