Design and structural analysis of hairpin-TFO for transcriptional activation of genes in S. cerevisiae.
Triplex forming oligonucleotides (TFOs) have the potential to modulate gene expression. While most of the experiments are directed towards triplex mediated inhibition of gene expression the strategy potentially could be used for gene specific activation. In an attempt to design a strategy for gene specific activation in vivo applicable to a large number of genes we have designed a TFO based activator-target system which may be utilized in Saccharomyces cerevisiae or any other system where Gal4 protein is ectopically expressed. The total genome sequence of Saccharomyces cerevisiae and expression profiles were used to select the target genes with upstream poly (pu/py) sequences. We have utilized the paradigm of Gal4 protein and its binding site. We describe here the selection of target genes and design of hairpin-TFO including the targeting sequences containing polypurine stretch found in the upstream promoter regions of weakly expressed genes. We demonstrate, the formation of hairpin-TFO, its binding to Gal4 protein, its ability to form triplex with the target duplex in vitro, the effect of polyethylenimine on complex formation and discuss the implication on in vivo transcription activation.
D001483:Base Sequence; D001665:Binding Sites; D015825:Chromosomes, Fungal / Q000235:genetics; D002942:Circular Dichroism; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005800:Genes, Fungal; D008954:Models, Biological; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D011094:Polyethyleneimine / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011094:Polyethyleneimine