Figure 1.

Experimental scheme to investigate induction of intrachromosomal recombination by TFOs. LTK⁻ cells carrying, at a single chromosomal locus, two mutant copies of the TK gene as direct repeats flanking a polypurine third-strand binding site were used to test the ability of transfected or microinjected TFOs to promote recombination. A purine-rich oligonucleotide of length 30 (AG30) was designed to form a triple helix in the antiparallel triplex motif at the G-rich target site, as shown. As a control, Scr30, containing the same base composition but a scrambled sequence, was used. In some experiments, the AG30 and the SCR30 oligonucleotides were conjugated at their 5′ ends to 4′-hydroxymethyl-4,5′,8-trimethylpsoralen via the 4′-hydroxymethyl position. In this case, by formation of the triple helix, psoralen intercalation and photoaddition is targeted to the thymidines at the predicted duplex-triplex junction. Potential recombinants are identified as TK⁺ clones growing in selective HAT (1 × 10⁻⁴ M hypoxanthine/2 × 10⁻⁶ M aminopterin/1.6 × 10⁻⁵ M thymidine)-containing medium.