DNA Triple-Helix Formation at Physiologic pH and Temperature

Abstract

ABSTRACT

Oligonucleotides that form a triple helix with duplex DNA offer a novel way to site specifically regulate gene expression in vivo. Triple helices formed by homopyrimidine oligomers containing both cytosine and thymine are stabilized by acid pH and low temperature, and there is little information about triplex formation with these oligomers at both pH 7.5 and 37°C. Therefore, we examined the effect of changing various conditions on triplex formation at pH 7.5. A 30-mer oligonucleotide (composed of T and 5-methyl C) at submicromolar concentrations formed a triplex with its target duplex at pH 7.5 and 37°C. Association of the 30-mer oligomer with the duplex was slow, with complete association requiring about 1 h. At 37°C, a 21-mer oligomer bound weakly to the target duplex but both a 25-mer and the 30-mer readily formed a triplex. This relationship of triplex formation with length was temperature dependent, as at 25°C the 21-mer behaved similarly to the longer oligomers. Increasing spermine concentrations (from 0.2 to 1 mM) increased the amount of triplex formed. Spermine may be important only for the association of the oligomer to the duplex, since decreasing the spermine concentration after the triplex formed did not reduce the amount of triplex detected. At 1 mM spermine, formation of the triple-helical complex was very dependent on the concentration of KCl; increasing the KCl from 50 to 100 mM prevented triplex formation. However, the inhibitory effect of KCl could be abrogated by raising the spermine concentration to 2 mM. Our observations indicate that a triple helix can form under physiologic conditions but its formation is affected by several competing interactions.