Triplex targets in the human rhodopsin gene.
We have explored the application of triplex technology to the human rhodopsin gene, which encodes a G-protein-linked receptor involved in the genetic disorder autosomal dominant retinitis pigmentosa (ADRP). Our results support the hypothesis that most human genes contain high-affinity triplex sites and further refine the rules governing identification and successful targeting of triplex-forming oligonucleotides (TFOs) to these sites. Using a computer search for sites 15 nucleotides in length and greater than 80% purine, we found 143 distinct sites in the rhodopsin gene and comparable numbers of sites in several other human genes. By applying more stringent criteria, we selected 17 potential target sites in the rhodopsin gene, screened them with a plasmid binding assay, and found 8 that bound TFOs with submicromolar affinity (Kd = 10(-)9-10(-)7 M). We compared purine (GA) and mixed (GT) TFOs at each site, and found that GA-TFOs consistently bound with higher affinity, and were less sensitive to pyrimidine interruptions in the target strand. High G-content favored high-affinity binding; only sites with >54% G-content bound TFOs with Kd </= 10(-)8 M.
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
B D
BD
Perkins
J H
JH
Wilson
T G
TG
Wensel
K M
KM
Vasquez
D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000235:genetics; D012243:Rhodopsin / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity
D009841:Oligonucleotides; C051802:triplex DNA; D004247:DNA; D012243:Rhodopsin