1030. Pyrazolo[3,4-d]Pyrimidine Guanine Base Substitution in Triplex Forming Oligonucleotides Improves Target Specificity and Chromosomal Mutagenesis
Year: 2006
Type: article
Abstract: Triplex forming oligonucleotides (TFOs) can induce site-specific damage, repair and recombination in episomal and chromosomal targets. However, several limitations preclude their effective use for gene targeting at the present time. For example, purine-rich TFOs have reduced binding in physiologic levels of monovalent cations, especially K+, apparently due to self-association at the guanine N7. We have substituted a 30-mer purine-rich TFO with the base analog 7-deaza-8-aza-guanine (PPG), in which the N7 and C8 positions of the guanine ring are exchanged. We show here that the PPG- substituted TFOs have increased binding affinity to a polypurine target site in vitro, even in the presence of physiologic [K+]. As well, the PPG-substituted TFOs are more efficient at forming covalent crosslinks at a chromosomally integrated target site. Notably, TFOs containing PPG substitutions at every third guanine induced almost a four-fold increase in mutation frequency in a chromosomal reporter gene in mammalian cells, almost twice the rate as a TFO containing natural guanine. In addition, we are interested in using modified TFOs to target disease-related genes in mammalian cells; preliminary results indicate that PPG-substituted TFOs can target an endogenous intronic sequence of the beta-globin locus to promote gene correction. PPG modifications, therefore, can augment triplex specificity and stability, resulting in enhanced gene targeting and correction.
Source: Molecular Therapy
Institution: Yale University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
OpenAlex ID: W2007334494