Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0W0, Canada Received November 6, 1990; Revised Manuscript Received February 13, 1991 abstract: The stability of triplex DNA was investigated in the presence of the polyamines spermine and spermidine by four different techniques. First, thermal-denaturation analysis of poly [d (TC)]-poly [d (GA)] showed that at low ionic strength and pH 7, 3^ M spermine was sufficient to cause dismutation of all of the duplex to the triplex conformation. A 10-fold higher concentration of spermidine produced a similar effect. Second, the kinetics of the dismutation were measured at pH 5 in 0.2 M NaCl. The addition of 500/tM spermine increased the rate by at least 2-fold. Third, in 0.2 M NaCl, the mid-point of the duplex-to-triplex dismutation occurredat a pH of 5.8, but this was increased by nearly one pH unit in the presence of 500/uM spermine. Fourth, intermolecular triplexes can also form inplasmids that contain purine-pyrimidine inserts bythe addition of a single-stranded pyrimidine. This was readily demonstrated at pH 7.2 and 25 mM ionic strength in the presence of 100 jtM spermine or spermidine. In 0.2 M NaCl, however, 1 mM polyamine is required. Since, in the eucaryotic nucleus, the polyamine concentration is in the millimolar range, then appropriate purine-pyrimidine DNA sequences may favor the triplex con-formation invivo.

Triplex structures were first described over 20 years ago (Glaser & Gabbay, 1968; Morgan & Wells, 1968). In the more usual form, they consist of TAT and CG-C+ base triads in which the third pyrimidine strand winds up the major groove of an A-form helix with Hoogsteen pairing. This ne-cessitates protonation of one of the cytosines, and the two pyrimidine strands are antiparallel. Although no X-ray crystallographic structure is yet available, recent NMR studies support this simple model (Rajagopal & Feigon, 1989; de los Santos et al., 1989). These requirements tend to restricttriplex formation to polypurine-polypyrimidine sequences (pur-pyr DNA) although some mismatches can be tolerated (Hanvey et al., 1989; Griffin & Dervan, 1989). In addition, a low pH is generally required because of the protonated cytosine, which has a pKa of 4.5 in the free state. Thus synthetic DNAs such as poly [d (TC)]-poly [d (GA)] will dismutate to a triplex at pHs below 6 (Lee et al., 1979). Although this result seems to preclude a physiological role for triplexes, several lines of evidence are suggestive of their presence in eucaryotic cells. First, pur-pyr tracts, some of which are over 100 base pairs in length, represent up to 1% of certain eucaryotic genomes (Hoffman-Lieberman et al., 1986; Manor et al., 1988). Thus the concentration of potential triplex-forming sequences in the