EFFECT OF ADDITIONAL UNPAIRED BASES ON THE STABILITY OF 3-WAY DNA JUNCTIONS STUDIED BY FLUORESCENCE TECHNIQUES

Fluorescence melting experiments were carried but to determine the rel ative stability of three-way DNA junctions with and without extrahelic al adenine nucleotides in one strand at the branch point of the juncti on (i.e., A(n) bulges where n = 0, 1, 2, and 3). The oligonucleotides were labeled with chromophores at the 5' ends of the strands. The prog ress of the thermal denaturation was followed by monitoring the fluore scence intensities and anisotropies of the dyes and the fluorescence r esonance energy transfer between the two dyes. The results of the ther mal denaturation experiments are interpreted and discussed in terms of either two-state thermodynamic models or statistical models for the t hermal denaturation. The junctions all melt at the same temperature (a t equal concentrations) within the error of the T-m determination, reg ardless of the presence, or absence, of the bulge. It 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 ju nctions, all which have 10 base pairs in each arm, possess thermal den aturation characteristics similar to duplexes with 20 arms. This leads to the proposition that for these junctions an important molecular pa rameter 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 s trongly on the nucleotide sequence in the single-stranded region.