Triplex-mediated, in vitro targeting of psoralen photoadducts within the genome of a transgenic mouse.

Abstract

Light-activated psoralens can covalently modify DNA and are widely used to study nucleic acid secondary structure and mutagenesis. Sequence specificity can be added to the photoaddition reaction by attaching the psoralen to an oligonucleotide designed to recognize a double-stranded DNA binding site through formation of a triple helix. We have previously used this strategy to study targeted psoralen modification of a triplex binding site within the bacterial supF gene carried in viral genomes. In the present work we report the targeting of psoralen photoadducts in vitro to a specific site in the genome of a transgenic mouse. Both 10 base and 16 base oligonucleotide-psoralen conjugates were capable of sequence-specific modification of genomic mouse DNA, while a truncated 8 base conjugate was not. Light activation was necessary, and a dose dependence was demonstrated for target site modification and mutagenesis. The 10 base conjugate rapidly found its target, with sequence-specific binding occurring after just 10 min incubation in the presence of mouse DNA. The ability to target psoralen photoadducts within mammalian genomes may prove useful in the study of chromatin structure and DNA repair. Moreover, this work may lead to potential in vivo applications of targeted psoralen modification.