A series of inserts with oligopurine.oligopyrimidine mirror repeat sequences was investigated at the base pair level with specific chemical probes (OsO4 and diethylpyrocarbonate) to evaluate the in vitro existence of intramolecular triplexes. Two parent inserts in recombinant plasmids with (GAA)9 and (AG)12 sequences and three mutant inserts (containing transitions or transversions) revealed that base pair changes at one location affected the chemical reactivity 13 base pairs away. The specificity and nature of these reactions, as well as the thermal stability of the complexes, provide direct evidence for the existence of a triplex with a portion of the pyrimidine-rich strand folded back and Hoogsteen-paired in the major groove of the Watson-Crick duplex. The biological implications of this unorthodox DNA structure are discussed
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