GC-rich flanking tracts decrease the kinetics of intramolecular DNA triplex formation.

Abstract

The effect of the base composition of flanking sequences on DNA intramolecular triplex formation was investigated in negatively supercoiled plasmids. The rates of triplex formation at two oligopurine.oligopyrimidine inserts with interrupting sequences in plasmids containing AT- or GC-rich flanking sequences were compared as a function of temperature, pH, and negative superhelical density. The kinetics of the transition of linear B-DNA to triplex (also called H-DNA) were influenced by all of these factors; triplexes were formed slower in a GC-rich background than in an AT-rich background. However, at equilibrium, the same amounts of the triplexes in AT- or GC-rich contexts were formed, and the conformations adopted by (GAA)4TTCGC(GAA)4 showed the canonical intramolecular triplex as mapped with chemical probes. We propose that the GC-rich segments caused this effect by thermodynamically clamping the DNA inserts, since the dependence of kinetics on base composition disappeared in tetraalkylammonium ions which eliminate the dependence of helix-coil transitions on base composition. The dependence of the kinetics of intramolecular triplex formation on flanking sequences further strengthens the concept of the role of DNA as a dynamic participant in cellular events.