CONFORMATIONAL DISTRIBUTIONS OF A 4-WAY DNA JUNCTION REVEALED BY TIME-RESOLVED FLUORESCENCE RESONANCE ENERGY-TRANSFER

Conformational distributions of a four-way DNA junction have been exam ined by time-resolved fluorescence resonance energy transfer (FRET). A series of dye-labeled junctions were synthesized with donor (fluoresc ein) and acceptor (tetramethylrhodamine) dyes conjugated to the 5' ter mini of the duplex arms in all six pairwise combinations. The fluoresc ence decay of the donor in each junction was measured by time-correlat ed single-photon counting. The distributions of donor-acceptor (D-A) d istances present between each pair of arms were recovered from the don or decays using a continuous Gaussian distribution model. The overall geometry of the four-way junction defined by the six mean D-A distance s was consistent with a stacked-X structure, wherein pairs of duplex a rms associate to form two continuous domains. Large differences were o bserved in the widths of the D-A distance distributions, depending on which pair of arms were labeled with the donor and acceptor dyes. Dist ances measured along the stacking domains were characterized by relati vely narrow distributions, indicating that these domains were rigid, w hereas distances between stacking domains had broader distributions, r eflecting variability in the angle between the two domains. The distan ces described by broad distributions were overestimated by steady-stat e FRET measurements. These results suggest that an ensemble of stacked -X structures are present in solution, characterized by differences in the small angle between the stacking domains. Temperature and solvent effects on the recovered distribution widths provide an indication of flexibility in the four-way junction.