Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog.

Fumi Nagatsugi, Shigeki Sasaki, Paul S. Miller, Michael M. Seidman

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
JournalNucleic Acids Research
Volume31
Issue number6
StatePublished - Mar 15 2003
Externally publishedYes

Fingerprint

Site-Directed Mutagenesis
Nucleosides
Oligonucleotides
Cytosine
Mutation
Plasmids
Genetic Vectors
Genetic Engineering
Alkylation
DNA Repair
Genetic Therapy

ASJC Scopus subject areas

  • Genetics

Cite this

Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog. / Nagatsugi, Fumi; Sasaki, Shigeki; Miller, Paul S.; Seidman, Michael M.

In: Nucleic Acids Research, Vol. 31, No. 6, 15.03.2003.

Research output: Contribution to journalArticle

Nagatsugi, Fumi ; Sasaki, Shigeki ; Miller, Paul S. ; Seidman, Michael M. / Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog. In: Nucleic Acids Research. 2003 ; Vol. 31, No. 6.
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