Nucleic Acids Research, Vol 25, Issue 3 633-640, Copyright © 1997 by Oxford University Press
AF Faruqi, SH Krawczyk, MD Matteucci and PM Glazer
Triple helix formation by purine-rich oligonucleotides in the anti-
parallel motif is inhibited by physiological concentrations of potassium.
Substitution with 7-deazaxanthine (c7X) has been suggested as a strategy to
overcome this effect. We have tested this by examining triple helix
formation both in vitro and in vivo by a series of triple helix-forming
oligonucleotides (TFOs) containing guanine plus either adenine, thymine, or
c7X. The TFOs were conjugated to psoralen at the 5'end and were designed to
bind to a portion of the supF mutation reporter gene. Using in vitro gel
mobility shift assays, we found that triplex formation by the
c7X-substituted TFOs was relatively resistant to the presence of 140 mM K+.
The c7X-containing TFOs were also superior in gene targeting experiments in
mammalian cells, yielding 4- to 5-fold higher mutation frequencies in a
shuttle vector-based mutagenesis assay designed to detect mutations induced
by third strand- directed psoralen adducts. When the phosphodiester
backbone was replaced by a phosphorothioate one, the in vitro binding of
the c7X- TFOs was not affected, but the efficiency of in vivo triple helix
formation was reduced. These results indicate the utility of the c7X
substitution for in vivo gene targeting experiments, and they show that the
feasibility of the triplex anti-gene strategy can be significantly enhanced
by advances in nucleotide chemistry.
ARTICLES
Potassium-resistant triple helix formation and improved intracellular gene targeting by oligodeoxyribonucleotides containing 7-deazaxanthine
Department of Therapeutic Radiology, Yale University School of Medicine, PO Box 208040, New Haven, CT 06520-8040, USA.
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