CONFORMATIONAL FLEXIBILITY OF 3-WAY DNA JUNCTIONS CONTAINING UNPAIREDNUCLEOTIDES

Time-resolved fluorescence resonance energy transfer has been used to examine the global structure and conformational flexibility of three-w ay DNA junctions containing unpaired bases at the branch point. Three- way junctions were prepared with donor (fluorescein) and acceptor (tet ramethylrhodamine) dyes attached to the ends of different helical arms in various pairwise combinations. The time-resolved fluorescence deca y of the donor in each labeled junction was measured by time-correlate d single photon counting. The distributions of donor-acceptor (D-A) di stances present between each pair of labeled helices were recovered fr om analysis of the donor decay profiles using a Gaussian distribution model. The recovered D-A distance distributions reveal the mean distan ce between each pair of helices, as well as the range of distances tha t exists between each pair. For the junction lacking unpaired bases, t he three mean interarm distances are similar, indicating an extended s tructure. In addition, a relatively broad range of distances is presen t between each pair of helices, showing that the structure is flexible . The addition of unpaired bases causes the junction to fold into a di fferent structure, with one interarm distance being shorter than the o ther two. The change in overall geometry of the junction appears to be primarily due to the repositioning of one of the helices flanking the bulge. In bulged junctions containing unpaired thymine, cytosine, or adenine bases, the helix containing the 3' portion of the bulged stran d appears to undergo the greatest change in its' mean position relativ e to the other helices. In contrast, in the bulged junction containing unpaired guanine bases, the helix containing the 5' portion of the bu lged strand is displaced. In all bulged junctions, there is a wide ran ge of distances between the perturbed helix and the other two helices, indicating high mobility for the perturbed arm. These results indicat e that the overall structure and conformational flexibility of three-w ay DNA junctions are sensitive to the presence of unpaired bases at th e branch point of the junction and that the precise effect of a bulge depends on the nature of the unpaired bases.