Effect of additional unpaired bases on the stability of three-way DNA junctions studied by fluorescence techniques.

Abstract

Fluorescence melting experiments were carried out to determine the relative stability of three-way DNA junctions with and without extrahelical adenine nucleotides in one strand at the branch point of the junction (i.e., An bulges where n = 0, 1, 2, and 3). The oligonucleotides were labeled with chromophores at the 5' ends of the strands. The progress of the thermal denaturation was followed by monitoring the fluorescence intensities and anisotropies of the dyes and the fluorescence resonance energy transfer between the two dyes. The results of the thermal denaturation experiments are interpreted and discussed in terms of either two-state thermodynamic models or statistical models for the thermal denaturation. The junctions all melt at the same temperature (at equal concentrations) within the error of the Tm determination, regardless of the presence, or absence, of the bulge. It is suggested that the denaturation of the helical arms begins primarily at the free ends of the helical arms and proceeds toward the branch point. The junctions, all which have 10 base pairs in each arm, possess thermal denaturation characteristics similar to duplexes with 20 arms. This leads to the proposition that for these junctions an important molecular parameter that controls the stability of the junctions is the number of base pairs between neighboring arms. The melting profiles obtained by monitoring the tetramethylrhodamine fluorescence are found to depend strongly on the nucleotide sequence in the single-stranded region.