Design of a novel triple helix-forming oligodeoxyribonucleotide directed to the major promoter of the c-myc gene.
Altered expression of c-myc is implicated in pathogenesis and progression of many human cancers. Triple helix-forming oligonucleotides (TFOs) directed to a polypurine/polypyrimidine sequence in a critical regulatory region near the c-myc P2 promoter have been shown to inhibit c-myc transcription in vitro and in cells. However, these guanine-rich TFOs had moderate binding affinity and required high concentrations for activity. The 23 bp myc P2 sequence is split equally into AT- and GC-rich tracts. Gel mobility analysis of a series of short TFOs directed in parallel and anti-parallel orientation to the purine strand of each tract showed that only parallel CT and anti-parallel GT TFOs formed stable triplex on the AT- and GC-rich tracts, respectively. A novel full-length GTC TFO was designed to bind simultaneously in parallel and anti-parallel orientation to the polypurine strand. Gel-shift and footprinting assays showed that the new TFO formed a triple helix in physiological conditions with significantly higher affinity than an anti-parallel TFO. Protein-binding assays showed that 1 microM GTC TFO inhibited binding of nuclear transcription factors to the P2 promoter sequence. The novel TFO can be developed into a potent antigene agent, and its design strategy applied to similar genomic sequences, thus expanding the TFO repertoire.
McGuffie
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Department of Medicine, Division of Hematology/Oncology and Laboratory of Cancer Genomics, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA.
Catapano
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D020856:AT Rich Sequence; D001483:Base Sequence; D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry; D020862:GC Rich Sequence; D016259:Genes, myc; D006147:Guanine / Q000737:chemistry; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D013936:Thymidine / Q000737:chemistry; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D016271:Proto-Oncogene Proteins c-myc; C051802:triplex DNA; D006147:Guanine; D003596:Cytosine; D004247:DNA; D005090:Exodeoxyribonucleases; D013936:Thymidine