In vivo transcription of a progesterone-responsive gene is specifically inhibited by a triplex-forming oligonucleotide.
Oligonucleotides provide novel reagents for inhibition of gene expression because of their high affinity binding to specific nucleotide sequences. We describe a 38 base, single-stranded DNA that forms a triple helix or 'triplex' on progesterone response elements of a target gene. This triplex-forming oligonucleotide binds with a Kd = 100 nM at 37 degrees C and physiological pH, and blocks binding of progesterone receptors to the target. Furthermore, it completely inhibited progesterone receptor-dependent transcription in vitro. To approach in vivo conditions, triplex-forming oligonucleotides were tested in cell transfection studies. The derivation of the oligonucleotides with cholesterol enhanced their cellular uptake and nuclear concentration by at least four-fold. The cholesterol-derivatized triplex-forming oligonucleotide specifically inhibited transcription of the PRE-containing reporter gene in cells when applied to the medium at micromolar concentrations. This is the first demonstration of steroid-responsive gene inhibition by triplex formation and joins the growing body of evidence indicating that oligonucleotides have therapeutic potential.
Ing
N H
NH
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Beekman
J M
JM
Kessler
D J
DJ
Murphy
M
M
Jayaraman
K
K
Zendegui
J G
JG
Hogan
M E
ME
O'Malley
B W
BW
Tsai
M J
MJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002784:Cholesterol / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D066246:ErbB Receptors / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D000882:Haplorhini; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011374:Progesterone / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011980:Receptors, Progesterone / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D004277:DNA, Single-Stranded; D011980:Receptors, Progesterone; D011374:Progesterone; D004247:DNA; D002784:Cholesterol; D066246:ErbB Receptors