Abstract
Oligonucleotides can bind in the major groove of duplex DNA and form triple helices in a sequence-specific manner 1–4). Progress in elucidating the third strand binding code has raised the possibility of developing nucleic acids as sequence-specific reagents for research and possibly clinical applications. Oligonucleotide-mediated triplex formation has been shown to prevent transcription factor binding to promoter sites and to block mRNA synthesis in vitro and in vivo (5–8). Instead of using triplex formation to transiently block gene expression, however, we reasoned that it would be advantageous to use triple helix formation to target mutations to specific sites in selected genes in order to produce permanent, heritable changes in gene function and expression (9–11). In this approach, mutations are targeted to a selected site by linking the triplex-forming oligonucleotide to a mutagen so that the sequence specificity of the triplex formation can be imparted to the action of the mutagen (Fig. 1).
Strategy for triple helix-targeted mutagenesis. The 10-base triple helix-forming oligonucleotide is shown positioned above its target site in the supF gene (bp 167–176). The oligonucleotide is conjugated to a mutagen, 4′-hydroxymethyl-4,5′,8-trimethylpsoralen, which is targeted to intercalate between basepairs 166 and 167, as indicated by the arrow. Photoactivation of the psoralen generates adducts and thereby mutations at the targeted site.
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© 1996 Humana Press Inc.
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Glazer, P.M., Wang, G., Havre, P.A., Gunther, E.J. (1996). Targeted Mutagenesis Mediated by the Triple Helix Formation. In: Trower, M.K. (eds) In Vitro Mutagenesis Protocols. Methods In Molecular Medicine™, vol 57. Humana Press. https://doi.org/10.1385/0-89603-332-5:109
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DOI: https://doi.org/10.1385/0-89603-332-5:109
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