Molecular Scale Architecture: Engineered Three- And Four-Way Junctions (original) (raw)
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Stability and structure of three-way DNA junctions containing unpaired nucleotides
Nucleic Acids Research, 1991
Non-paired nucleotides stabilize the formation of threeway helical DNA junctions. Two or more unpaired nucleotides located in the junction region enable oligomers ten to fifteen nucleotides long to assemble, forming conformationally homogeneous junctions, as judged by native gel electrophoresis. The unpaired bases can be present on the same strand or on two different strands. Up to five extra bases on one strand have been tested and found to produce stable junctions. The formation of stable structures is favored by the presence of a divalent cation such as magnesium and by high monovalent salt concentration. The order-disorder transition of representative threeway junctions was monitored optically in the ultraviolet and analyzed to quantify thermodynamically the stabilization provided by unpaired bases in the junction region. We report the first measurements of the thermodynamics of adding an unpaired nucleotide to a nucleic acid three-way junction. We find that AAG0(371C) = + 0.5 kcal/mol for increasing the number of unpaired adenosines from two to three. Three-way junctions having reporter arms 40 base-pairs long were also prepared. Each of the three reporter arms contained a unique restriction site 15 base-pairs from the junction. Asymmetric complexes produced by selectively cleaving each arm were analyzed on native gels. Cleavage of the double helical arm opposite the strand having the two extra adenosines resulted in a complex that migrated more slowly than complexes produced by cleavage at either of the other two arms. It is likely that the strand containing the unpaired adenosines is kinked at an acute angle, forming a Yshaped, rather than a T-shaped junction.
The Stereochemistry of a Four-Way DNA Junction: a Theoretical Study
Nucleic acids research, 1990
The stereochemical conformation of the four-way helical junction in DNA (the Holliday junction; the postulated central intermediate of genetic recombination) has been analysed, using molecular mechanical computer modelling. A version of the AMBER program package was employed, that had been modified to include the influence of counterions and a global optimisation procedure. Starting from an extended planar structure, the conformation was varied in order to minimise the energy, and we discuss three structures obtained by this procedure. One structure is closely related to a square-planar cross, in which there is no stacking interaction between the four double helical stems. This structure is probably closely similar to that observed experimentally in the absence of cations. The remaining two structures are based on related, yet distinct, conformations, in which there is pairwise coaxial stacking of neighbouring stems. In these structures, the four DNA stems adopt the form of two quasi-continuous helices, in which base stacking is very similar to that found in standard B-DNA geometry. The two stacked helices so formed are not aligned parallel to each other, but subtend an angle of approximately 60°. The strands that exchange between one stacked helix and the other are disposed about the smaller angle of the cross (i.e. 60° rather than 120°), generating an approximately antiparallel alignment of DNA sequences. This structure is precisely the stacked X-structure proposed on the basis of experimental data. The calculations indicate distortions from standard B-DNA conformation that are required to adopt the stacked X-structure; a widening of the minor groove at the junction, and reorientation of the central phosphate groups of the exchanging strands. An important feature of the stacked X-structure is that it presents two structurally distinct sides. These may be recognised differently by enzymes, providing a rationalisation for the points of cleavage by Holliday resolvases.
Stereospecific Effects Determine the Structure of a Four-Way DNA Junction
Chemistry & Biology, 2005
square structure involves a lowering of symmetry from four-to twofold. There are therefore two distinct con-Taekjip Ha, 2 David G. Norman, 1 formers of the structure that are equivalent aside from and David M.J. Lilley 1, * the nucleotide sequence. This is illustrated for the junc-1 Cancer Research UK Nucleic Acid Structure tion shown schematically in Figure 1A. The four arms Research Group are sequentially labeled B, H, R, and X. These can un-MSI/WTB Complex dergo coaxial stacking in two alternative ways, either The University of Dundee based on H stacking on B (and therefore R on X) in the Dundee DD1 5EH isoI structure or B stacking on X (isoII). The component United Kingdom strands of the stacked X structure divide into two 2 Physics Department classes that interconvert on switching from the isoI to University of Illinois at Urbana-Champaign the isoII structure. The continuous strands (b and r in 1110 West Green Street the isoI structure, drawn black) run the full length of the Urbana, Illinois 61801 coaxially paired helices, while the exchanging strands (h and x in isoI) cross between the stacks, forming an exchange region at the center of the junction. In the Summary isoII structure, the b and r strands become the exchanging strands, while h and x are now continuous. A Conversion of a centrally located phosphate group to single junction can sample both stacking conformers, an electrically neutral methyl phosphonate in a fourwith continual conformational exchange between way DNA junction can exert a major influence on its structures. First suggested by experiments in bulk soluconformation. However, the effect is strongly depention [19, 20], this has recently been demonstrated dident on stereochemistry. Substitution of the proR rectly by single-molecule FRET spectroscopy [21, 22]. oxygen atom by methyl leads to conformational tran-Electrostatic forces are extremely important in the sition to the stacking conformer that places this folding of the four-way DNA junction, as indicated by phosphate at the point of strand exchange. By conthe critical role for metal ions in the folding process. trast, corresponding modification of the proS oxygen The formation of the stacked X structure requires either destabilizes this conformation of the junction. Singledivalent metal ions [3, 5] or very high concentrations molecule analysis shows that both molecules are in of monovalent ions [10, 23], and the rate of conformer a dynamic equilibrium between alternative stacking exchange is strongly dependent on salt concentration conformers, but the configuration of the methyl phos-[21, 22]. We have recently studied the role of phosphate phonate determines the bias of the conformational charge using substitution by electrically neutral methyl equilibrium. It is likely that the stereochemical enviphosphonates [24]. We found that if the central phosronment of the methyl group affects the interaction phate groups of the exchanging strands were rendered with metal ions in the center of the junction. neutral by substitution, the resulting junction was substantially folded into the stacked X structure without
Refinement of the solution structure of a branched DNA three-way junction
Biophysical Journal, 1995
We have refined the structure of the DNA Three-Way Junction complex, TWJ-TC, described in the companion paper by quantitative analysis of two 2D NOESY spectra (mixing times 60 and 200 ms) obtained in D20 solution. NOESY crosspeak intensities extracted from these spectra were used in two kinds of refinement procedure: 1) distance-restrained energy minimization (EM) and molecular dynamics (MD) and 2) full relaxation matrix back calculation refinement. The global geometry of the refined model is very similar to that of a published, preliminary model . Two of the helical arms of the junction are stacked. These are Helix 1, defined by basepairs S1-G1/S3-C12 through Sl-C5/S3-G8 and Helix 2, which comprises basepairs S1-C6/S2-G5 through S1-G1O/S2-G1. The third helical arm (Helix 3), comprised of basepairs S2-C6/S3-G5 through S2-Cl 0/S3-G1 extends almost perpendicularly from the axis defined by Helices 1 and 2. The bases S1-C5 and Si -C6 of Strand 1 are continuously stacked across the junction region. The conformation of this strand is close to that of B-form DNA along its entire length, including the S1 -C5 to Si -C6 dinucleotide step at the junction. The two unpaired bases S3-T6 and S3-C7 lie outside of the junction along the minor groove of Helix 1 and largely exposed to solvent. Analysis of the refined structure reveals that the glycosidic bond of S3-T6 exists in the syn conformation, allowing the methyl group of this residue to contact the hydrophobic surface of the minor groove of Helix 1, at S3-G11. The helical parameters of the three helical arms of the structure exhibit only weak deviations from typical values for right-handed B-form DNA. Unusual dihedral angles are only observed for the sugarphosphate backbone joining the "hinge" residues, S2-G5 and S2-C6, and S3-G5 through S3-G8. The glycosidic bond of S3-G8 also lies within the syn range, allowing favorable Watson-Crick base-pairing interactions with Si -C5. The stability of this structure was checked in 39 ps molecular dynamic simulation at 330 K in water. The structure of TWJ-TC retained the geometrical features mentioned above at the end of the simulation period. The final R(1/6)-factor of the refined structure is 5%.
Construction, Analysis, Ligation, and Self-Assembly of DNA Triple Crossover Complexes
Journal of the American Chemical Society, 2000
This paper extends the study and prototyping of unusual DNA motifs, unknown in nature, but founded on principles derived from biological structures. Artificially designed DNA complexes show promise as building blocks for the construction of useful nanoscale structures, devices, and computers. The DNA triple crossover (TX) complex described here extends the set of experimentally characterized building blocks. It consists of four oligonucleotides hybridized to form three double-stranded DNA helices lying in a plane and linked by strand exchange at four immobile crossover points. The topology selected for this TX molecule allows for the presence of reporter strands along the molecular diagonal that can be used to relate the inputs and outputs of DNA-based computation. Nucleotide sequence design for the synthetic strands was assisted by the application of algorithms that minimize possible alternative base-pairing structures. Synthetic oligonucleotides were purified, stoichiometric mixtures were annealed by slow cooling, and the resulting DNA structures were analyzed by nondenaturing gel electrophoresis and heat-induced unfolding. Ferguson analysis and hydroxyl radical autofootprinting provide strong evidence for the assembly of the strands to the target TX structure. Ligation of reporter strands has been demonstrated with this motif, as well as the self-assembly of hydrogen-bonded two-dimensional crystals in two different arrangements. Future applications of TX units include the construction of larger structures from multiple TX units, and DNA-based computation. In addition to the presence of reporter strands, potential advantages of TX units over other DNA structures include space for gaps in molecular arrays, larger spatial displacements in nanodevices, and the incorporation of well-structured out-of-plane components in two-dimensional arrays.
Nucleic Acids Research, 1995
Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotldes demonstrate that unpaired bases at the branch point stabilize three-and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang.Y.L, Mueller.J.E., Kemper.B. and Seeman.N.C. (1991) Biochemistry, 30, 5667-5674].