The ligation and flexibility of four‐arm DNA junctions

Four‐arm DNA branched junctions are stable analogs of Holliday recombination intermediates, constructed from oligonucleotides. The conformational flexibility of junctions can be estimated by ligating them together and determining the set of closed macrocyclic products that are obtained among the linked units. We have performed a series of these experiments, using pairs of sticky ends that flank each of the six angles of a four‐arm junction. In every case, the ligated junctions are separated by 20 nucleotide pairs, about two turns of DNA. All expected short linear products, starting with dimers, are observed for all ligations. All ligations result in a macrocyclic series that begins with trimers. Thus, over the time scale of these reactions, the arms of this junction can form angles as low as 60°. The response of this junction to torsional stress has been tested in a companion experiment. A smaller version of this same four‐arm junction has been oligomerized so that successive junctions are separated by 16 nucleotide pairs, approximately 1.5 turns of DNA. If junctions were as rigid as linear duplex DNA, this system would not be expected to form macrocycles until the continuous chain approaches the Shore–Baldwin limit, ca. 160 base pairs. However, macrocyclic closure is observed in a regular ligation ladder, starting from tetramers. Model building suggests that the most likely explanation for the observed closure is that the junction adopts two different conformations, which bend the continuous strand toward opposite grooves. The junction structures formed by these ligations represent fluctuations from equilibrium structures.