Binding of triple helix forming oligonucleotides to sites in gene promoters.
A class of triplex-forming oligodeoxyribonucleotides (TFOs) is described that can bind to naturally occurring sites in duplex DNA at physiological pH in the presence of magnesium. The data are consistent with a structure in which the TFO binds in the major groove of double-stranded DNA to form a three-stranded complex that is superficially similar to previously described triplexes. The distinguishing features of this class of triplex are that TFO binding apparently involves the formation of hydrogen-bonded G.GC and T.AT triplets and the TFO is bound antiparallel with respect to the more purine-rich strand of the underlying duplex. Triplex formation is described for targets in the promoter regions of three different genes: the human c-myc and epidermal growth factor receptor genes and the mouse insulin receptor gene. All three sites are relatively GC rich and have a high percentage of purine residues on one strand. DNase I footprinting shows that individual TFOs bind selectively to their target sites at pH 7.4-7.8 in the presence of millimolar concentrations of magnesium. Electrophoretic analysis of triplex formation indicates that specific TFOs bind to their target sites with apparent dissociation constants in the 10(-7)-10(-9) M range. Strand orientation of the bound TFOs was confirmed by attaching eosin or an iron-chelating group to one end of the TFO and monitoring the pattern of damage to the bound duplex DNA. Possible hydrogen-bonding patterns and triplex structures are discussed.
Durland
R H
RH
Center for Biotechnology, Baylor College of Medicine, Woodlands, Texas 77381.
Kessler
D J
DJ
Gunnell
S
S
Duvic
M
M
Pettitt
B M
BM
Hogan
M E
ME
D000818:Animals; D001483:Base Sequence; D003850:Deoxyribonuclease I / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics; D016259:Genes, myc; D006801:Humans; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D011972:Receptor, Insulin / Q000235:genetics
D009841:Oligonucleotides; D016271:Proto-Oncogene Proteins c-myc; D066246:ErbB Receptors; D011972:Receptor, Insulin; D003850:Deoxyribonuclease I