Triplex targeting of a native gene in permeabilized intact cells: Covalent modification of the gene for the chemokine receptor CCR5

A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5′-end of the purine motif TFO modified a guani...

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Bibliographic Details
Published in:Nucleic acids research 1998-03, Vol.26 (5), p.1324-1328
Main Authors: Belousov, Evgeniy S., Afonina, Irina A., Kutyavin, Igor V., Gall, Alexander A., Reed, Michael W., Gamper, Howard B., Wydro, Robert M., Meyer, Rich B.
Format: Article
Language:English
Subjects:
Alkylating Agents
Base Sequence
Berberine Alkaloids
Cell Line
DNA - chemistry
DNA - genetics
Gene Targeting
Guanine - chemistry
Humans
Intercalating Agents
Mechlorethamine - chemistry
Molecular Sequence Data
Molecular Structure
Mutagenesis, Site-Directed
Nucleic Acid Conformation
Oligodeoxyribonucleotides - chemistry
Oligodeoxyribonucleotides - genetics
Polymerase Chain Reaction
Receptors, CCR5 - genetics
Transfection
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Summary:A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5′-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the triplex-stabilizing intercalator coralyne. This site-directed modification of a native chromosomal gene in intact human cells under conditions where many limitations of triplex formation have been partially addressed underscores the potential of this approach for gene control via site-directed mutagenesis.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/26.5.1324