Nucleic Acids Research, 2003, Vol. 31, No. 6 e31
© 2003 Oxford University Press
Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog
1 Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka, CREST (JST), Japan, 2 Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA and 3 Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA
*To whom correspondence should be addressed. Tel: +1 410 558 8565; Fax: +1 410 558 8157; Email: seidmanm{at}grc.nia.nih.gov
The specific recognition of homopurine–homo pyrimidine regions in duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Alkylation of nucleobases with functionalized TFOs would have the potential for site-directed mutagenesis. Recently, we demonstrated that a TFO bearing 2-amino-6-vinylpurine derivative, 1, achieves triplex-mediated reaction with high selectivity toward the cytosine of the G-C target site. In this report, we have investigated the use of this reagent to target mutations to a specific site in a shuttle vector plasmid, which replicates in mammalian cells. TFOs bearing 1 produced adducts at the complementary position of 1 and thereby introduced mutations at that site during replication/repair of the plasmid in mammalian cells. Reagents that produce covalent cytosine modifications are relatively rare. These TFOs enable the preparation of templates carrying targeted cytosine adducts for in vitro and in vivo studies. The ability to target mutations may prove useful as a tool for studying DNA repair, and as a technique for gene therapy and genetic engineering.
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