DNA structural changes induced by intermolecular triple helix formation

DNase I footprints of intermolecular DNA triplexes are often accompanied by enhanced cleavage at the 3&prime;-end of the target site at the triplex&ndash;duplex junction. We have systematically studied the sequence dependence of this effect by examining oligonucleotide binding to sites flanked by each base in turn. For complexes with a terminal T.AT triplet, the greatest enhancement is seen with ApC, followed by ApG and ApT, with the weakest enhancement at ApA. Similar DNase I enhancements were observed for a triplex with a terminal C+.GC triplet, though with little difference between the different GpN sites. Enhanced reactivity to diethylpyrocarbonate was observed at As that flank the triplex&ndash;duplex junction at&nbsp;AAA or&nbsp;AAC but not&nbsp;AAG or&nbsp;AAT. Fluorescence melting experiments demonstrated that the flanking base affected the stability with a 4 &deg;C difference in&nbsp;Tm&nbsp;between a flanking C and G. Sequences that produced the strongest enhancement correlated with those having the lower thermal stability. These results are interpreted in terms of oligonucleotide-induced changes in DNA structure and/or flexibility.</p

The reaction mechanism of Fokl excludes the possibility of targeting zinc finger nucleases to unique DNA sites

The FokI endonuclease is a monomeric protein with discrete DNA-recognition and catalytic domains. The latter has only one active site so, to cut both strands, the catalytic domains from two monomers associate to form a dimer. The dimer involving a monomer at the recognition site and another from free solution is less stable than that from two proteins tethered to the same DNA. FokI thus cleaves DNA with two sites better than one-site DNA. The two sites can be immediately adjacent, but they can alternatively be many hundreds of base pairs apart, in either inverted or repeated orientations. The catalytic domain of FokI is often a component of zinc finger nucleases. Typically, the zinc finger domains of two such nucleases are designed to recognize two neighbouring DNA sequences, with the objective of cutting the DNA exclusively between the target sequences. However, this strategy fails to take account of the fact that the catalytic domains of FokI can dimerize across distant sites or even at a solitary site. Additional copies of either target sequence elsewhere in the chromosome must elicit off-target cleavages.</jats:p

Stability of the different arms of a DNA tetrahedron and its interaction with a minor groove ligand

DNA strands can be designed to assemble into stable three-dimensional structures, based on Watson-Crick base pairing rules. The simplest of these is the DNA tetrahedron that is composed of four oligonucleotides. We have re-designed the sequence of a DNA tetrahedron so that it contains a single (AATT) binding site for the minor groove binding ligand Hoechst 33258. We examined the stability of this structure by placing fluorescent groups within each of its edges and have shown that all the edges melt at the same temperature in the absence of the ligand. The minor groove ligand still binds to its recognition sequence within the tetrahedron and increases the melting temperature of the folded complex. This ligand-induced stabilisation is propagated into the adjacent helical arms and the tetrahedron melts as a single entity in a cooperative fashion

Functionalizing designer DNA crystals with a triple-helical veneer.

DNA is a very useful molecule for the programmed self-assembly of 2D and 3D nanoscale objects.1 The design of these structures exploits Watson-Crick hybridization and strand exchange to stitch linear duplexes into finite assemblies.2-4 The dimensions of these complexes can be increased by over five orders of magnitude through self-assembly of cohesive single-stranded segments (sticky ends).5, 6 Methods that exploit the sequence addressability of DNA nanostructures will enable the programmable positioning of components in 2D and 3D space, offering applications such as the organization of nanoelectronics,7 the direction of biological cascades,8 and the structure determination of periodically positioned molecules by X-ray diffraction.9 To this end we present a macroscopic 3D crystal based on the 3-fold rotationally symmetric tensegrity triangle3, 6 that can be functionalized by a triplex-forming oligonucleotide on each of its helical edges

Stabilisation of self-assembled DNA crystals by triplex-directed photo-cross-linking

The tensegrity triangle is a robust DNA motif that can self-assemble to generate macroscopic three-dimensional crystals. However, the stability of these crystals is dependent on the high ionic conditions used for crystal growth. Here we demonstrate that a triplex-forming oligonucleotide can be used to direct the specific intercalation, and subsequent photo-cross-linking, of 4,5′,8-trimethylpsoralen to single or multiple loci within or between the tiles of the crystal. Cross-linking between the tiles of the crystal improves their thermal stability. Such an approach is likely to facilitate the removal of crystals from their mother liquor and may prove useful for applications that require greater crystal stability