Selectivity of polyamines in triplex DNA stabilization.

Abstract

Triplex DNA has been recently studied as an anti-gene strategy to suppress the transcription of specific genes. A major challenge in this area is to stabilize triplex DNA at physiological conditions. We studied the effects of putrescine, spermidine, spermine, their synthetic homologs, and their acetyl derivatives on triple- and double-stranded structures formed from poly(dA) and poly(dT) by measuring their respective melting temperatures. In the presence of polyamines, the absorbance (A) versus temperature (T) profile showed two transitions: Tm1, corresponding to triplex-->duplex+single-stranded DNA, and Tm2, corresponding to duplex melting. In the presence of 0.5 mM putrescine, Tm1 and Tm2 were 44.8 and 71 degrees C, respectively, in 10 mM sodium cacodylate buffer (pH 7.2). In contrast, triplex DNA was not detectable when the A versus T profile of the polynucleotides was monitored in the absence of putrescine. Tm2 was also lower (55 degrees C) in the absence of putrescine. With 2.5 microM spermidine or 0.1 microM spermine, Tm1 values were 42.8 and 54.4 degrees C and Tm2 values were 65 and 82 degrees C, respectively. As the concentration of polyamine was increased, the difference between Tm1 and Tm2 decreased, and both melting transitions merged into one Tm, corresponding to the melting of triplex DNA to single strands. In a series of putrescine homologs, H2N (CH2)nNH2 where n = 2-6 (n = 4 for putrescine), H2N(CH2)3NH2 was the most effective diamine to stabilize the poly(dA).2poly(dT) triplex. At 10 mM concentration, diaminopropane stabilized the triplex DNA such that the Tm1 was 10 degrees C higher than that in the presence of an equimolar concentration of putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)