Nucleic Acids Research, 2002, Vol. 30, No. 12 2701-2709
© 2002 Oxford University Press
Design of a novel triple helix-forming oligodeoxyribonucleotide directed to the major promoter of the c-myc gene
Department of Medicine, Division of Hematology/Oncology and Laboratory of Cancer Genomics, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA
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 µM 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.
* To whom correspondence should be addressed at: Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas Street, PO Box 250955, Charleston, SC 29425, USA. Tel: +1 843 792 6648; Fax: +1 843 792 3200; Email: catapanc{at}musc.edu
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