The ligation and flexibility of four‐arm DNA junctions

Abstract

SynopsisFour-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 dimem, 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 60O. 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.

Algorithmic Self-Assembly of DNA

“…Design components for crystals must have definable intermolecular interactions and must be rigid enough to prevent the formation of ill-defined aggregates (Liu et al 1994). Branched DNA molecules with sticky ends appear to be promising for macromolecular crystal design (Seeman 1982), because their intermolecular interactions can be programmed through sticky ends (Cohen et al 1973) that associate to form B-DNA (Qiu et al 1997); however, studies of three-and four-arm junctions reveal that the angles flanking their branchpoints are flexible (Ma et al 1986;Petrillo et al 1988).…”

Structure and dynamics of supercoil-stabilized DNA cruciforms

“…The ability of cruciforms and four-way DNA junctions (Cooper & Hagerman, 1987;Lilley & Clegg, 1993;Seeman & Kallenbach, 1994;Lilley, 1997;Srinivasan & Olson, 1994;Eis & Millar, 1993;Petrillo et al, 1988) to adopt different conformations might be functionally important. These DNA structures are the targets for different types of proteins such as recombination enzymes (Parsons et al, 1995;Yu et al, 1997), topoisomerases (Roca et al, 1993), and architectural proteins such as HMG1 and histones H1 and H5 (Timsit & Moras, 1996), so that the conformational¯exibility of DNA is an important factor for accommodating such a plethora of proteins.…”

Experiments in structural DNA nanotechnology: arrays and devices

“…Single-branched junctions, such as the HJ structure in Figure 1c are relatively flexible. 8,18 Fortunately, the DX molecule is considerably more rigid, apparently stiffer than even double helical DNA. 19 The TX and PX molecules appear to share this rigidity 20 Consequently, it has been possible to use these molecules as the building blocks of both arrays and devices.…”