The effects of interruptions in the homopurine bias and the G+C content of the homopurine.homopyrimidine (pur.pyr) sequences on intramolecular triplex formation and stability in supercoiled plasmids were evaluated. In addition, the interconversion of triplex and duplex, after altering the stabilizing factors (low pH or supercoiling), was studied. We conclude: (a) a 42-base pair pur.pyr sequence with three consecutive interruptions does not form a large triplex with three unpaired nucleotides in

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... the stem. Instead, a mixture of two smaller (27- and 28-nucleotide) triplexes forms. (b) A 28-nucleotide sequence with a single interruption forms a triplex with one unpaired nucleotide in the stem. This interruption causes the triplex to be 7 degrees C less thermostable and requires more superhelical energy for formation than the control triplex. (c) As the G+C content of a pur.pyr sequence increases, the thermostability of the triplex increases and the triplex requires less supercoiling for formation. (d) The interconversion between duplex and triplex is fast. After negative supercoiling is removed, all triplex becomes duplex in about 3 min. When the pH is shifted from 8.0 to 5.2, the conversion of duplex to triplex in a negatively supercoiled plasmid is complete in less than 2 min. Hence, these kinetic properties are consistent with important biological roles for triplexes. In summary, the results from both this and the accompanying paper show that a substantial amount of sequence imperfections is tolerated for triplex formation and stability.