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Biomedical subjects

N C Seeman

Publications and source records attributed to N C Seeman.

At least 19 recordsLinked to original sources

DNA junctions, antijunctions, and mesojunctions.

Antijunctions and mesojunctions are new classes of multistranded DNA complexes. They represent a generalization of DNA branched junction complexes, such as the Holliday recombination intermediate. Each strand of a conventional branched junction participates in two different double helices, and this is also true for mesojunctions and antijunctions. The helix axes of conventional branched junction complexes may be drawn to converge at a point, but this convergence occurs for lines drawn perpendicular to the helix axes of antijunctions. Mesojunctions are complexes that mix these features of junctions and antijunctions. Antijunction complexes require an even number of strands. We have synthesized the mesojunction containing three strands, the two mesojunctions containing four strands, and the antijunction containing four strands; we compare them with branched junctions containing three and four strands, derived by permutations of the same sequences. Each double helix is designed to contain 1.5 turns of DNA. A tendency to oligomerize makes it difficult to capture antijunctions and mesojunctions in stable discrete complexes, in contrast to conventional branched junctions. For both three-strand and four-strand complexes, Tm is highest for conventional branched junctions. Ferguson analysis indicates similarities in the occluded surface area of junctions, antijunctions, and one four-strand mesojunction, but the other four-strand mesojunction has a much lower apparent surface area. Hydroxyl radical cleavage patterns suggest that the four-strand antijunction and the low-surface-area four-strand mesojunction form stacking domains, analogous to the behavior of conventional branched junctions. These new structures are related to replicational and recombinational intermediates and to single-stranded nucleic acid knots.

Base Sequence

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.

Base Composition

Parallel and antiparallel Holliday junctions differ in structure and stability.

Two Holliday junction analogs, JA and JP, containing identical base-paired arms have been constructed from oligonucleotides. The former is constrained to adopt an antiparallel Sigal-Alberts structure, and the latter a parallel structure, by means of single strand d(T)9 tethers. We evaluate here the free energy difference between JA and JP using two different methods. One is a direct measurement of the ratio of the equilibrium constants for formation of branched structures from intact duplexes using one labeled strand and a competition assay. The second method estimates the difference in stability from the difference in thermal denaturation temperatures of JA and JP, using urea to shift the tm of the complexes. Both methods reveal a small free energy difference between the two complexes: JA is more stable than JP by -1.1(+/- 0.4) kcal (mol junction)-1, at 25 degrees C, 5 mM-Mg2+, from the first method, and by -1.6(+/- 0.3) kcal (mol junction)-1, according to the second. DNase I and the resolvase, endonuclease I from phage T7, cleave JA differently from JP in the vicinity of the branch, indicating that the structures of these two models differ at this site. Diethyl pyrocarbonate also reveals a difference in the major grooves. Comparison of the scission patterns of JA and JP by the reactive chemical probes methidium-propyl-EDTA..Fe(II), [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2,[(OP)2Cu(I)], indicates that in both cases the branch point is a site of enhanced binding for drugs, as it is in the untethered four-arm junction containing the same core sequence at the branch.

Base Composition

Assembly and characterization of five-arm and six-arm DNA branched junctions.

DNA branched junctions have been constructed that contain either five arms or six arms surrounding a branch point. These junctions are not as stable as junctions containing three or four arms; unlike the smaller junctions, they cannot be shown to migrate as a single band on native gels when each of their arms contains eight nucleotide pairs. However, they can be stabilized if their arms contain 16 nucleotide pairs. Ferguson analysis of these junctions in combination with three-arm and four-arm junctions indicates a linear increase in friction constant as the number of arms increases, with the four-arm junction migrating anomalously. The five-arm junction does not appear to have any unusual stacking structure, and all strands show similar responses to hydroxyl radical autofootprinting analysis. By contrast, one strand of the six-arm junction shows virtually no protection from hydroxyl radicals, suggesting that it is the helical strand of a preferred stacking domain. Both junctions are susceptible to digestion by T4 endonuclease VII, which resolves Holliday junctions. However, the putative helical strand of the six-arm junction shows markedly reduced cleavage, supporting the notion that its structure is largely found in a helical conformation. Branched DNA molecules can be assembled into structures whose helix axes form multiply connected objects and networks. The ability to construct five-arm and six-arm junctions vastly increases the number of structures and networks that can be built from branched DNA components. Icosahedral deltahedra and 11 networks with 432 symmetry, constructed from Platonic and Archimedean solids, are among the structures whose construction is feasible, now that these junctions can be made.

Base Sequence

Synthesis from DNA of a molecule with the connectivity of a cube.

A principal goal of biotechnology is the assembly of novel biomaterials for analytical, industrial and therapeutic purposes. The advent of stable immobile nucleic acid branched junctions makes DNA a good candidate for building frameworks to which proteins or other functional molecules can be attached and thereby juxtaposed. The addition of single-stranded 'sticky' ends to branched DNA molecules converts them into macromolecular valence clusters that can be ligated together. The edges of these frameworks are double-helical DNA, and the vertices correspond to the branch points of junctions. Here, we report the construction from DNA of a covalently closed cube-like molecular complex containing twelve equal-length double-helical edges arranged about eight vertices. Each of the six 'faces' of the object is a single-stranded cyclic molecule, doubly catenated to four neighbouring strands, and each vertex is connected by an edge to three others. Each edge contains a unique restriction site for analytical purposes. This is the first construction of a closed polyhedral object from DNA.

Base Sequence

The electrophoretic properties of a DNA cube and its substructure catenanes.

Electrophoretic properties have been measured for a DNA molecule whose helix axes have the connectivity of a cube. This molecule is a topologically bonded complex of six cyclic 80-mer molecules, in which each cycle corresponds to a face of the cube. Each cyclic molecule is doubly catenated to each of its four neighbors. Substructures of this molecule include a 5-cycle structure lacking one strand, two topoisomers of 4-cycle structures and two topoisomers of 3-cycle structures. One 4-cycle structure is a cyclic belt around the cube, lacking a top and a bottom, whereas the other lacks two catenated strands, such as the top and the front. One 3-cycle structure is a linear belt of three cycles, and the other corresponds to the three cycles that surround a corner. Each of these molecules is separable from the others under appropriate gel conditions. We have measured mobilities and calculated Ferguson plots for each of these molecules on polyacrylamide gels under both native and denaturing conditions. The measurements have been made with 1.25, 2.5, and 5% crosslinking of the gels. The data show that the higher-symmetry 3-cycle and 4-cycle structures migrate more slowly than their lower-symmetry isomers, under conditions where their Ferguson plots are parallel.

DNA

Construction of three-dimensional stick figures from branched DNA.

Stable DNA branched junction molecules can be used as the building blocks for stick-figures in which the edges are double-helical DNA and the vertices correspond to the branch points of the junctions. Sticky-ended cohesion is used to direct the association of individual branched complexes. The sequences of these molecules are assigned by a sequence-symmetry minimization procedure. Successful ligation experiments include the oligomerization of individual three-arm and four-arm junctions, the assembly of a quadrilateral from four junctions with different sticky ends, and the recent construction of a molecule with the connectivity of a cube. Possible applications include the assembly of molecular electronic devices, the formation of macromolecular-scale zeolites to host biological complexes for diffraction analysis, and the development of new catalysts.

Base Sequence

Characterization of a bimobile DNA junction.

We present here a chemical and enzymatic footprinting analysis of a branched DNA molecule formed from four complementary 50-mer strands. These strands are designed to form a stable junction, in which two steps of branch point migration freedom are possible. Exposure of the junction to Fe(II).EDTA shows protection of 3 or 4 residues in each strand at the branch, while two resolvase enzymes (endonuclease VII from phage T4 and endonuclease I from phage T7), cleave all four strand near the branch. Chemical footprinting of this junction using the reagents MPE.Fe(II) and (OP)2Cu(I) shows that the branch site is hyper-reactive to cutting induced by these probes as it is in an immobile four-arm junction. The effects involve more residues than in the immobile case. In the absence of divalent cations, the structure of the junction alters, sites of enhanced cleavage by MPE.Fe(II) and (OP)2Cu(I) disappear, and purines at the branch become reactive to diethyl pyrocarbonate. Our interpretation of these results is based on the properties of immobile junction analogs and their response to these probes. In the presence of Mg2+, the three migrational isomers coexist, each probably in the form of a 2-fold symmetric structure with two helical arms stacked.

Base Sequence

Resolution of Holliday junction analogs by T4 endonuclease VII can be directed by substrate structure.

Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction.

Base Sequence

Construction and analysis of parallel and antiparallel Holliday junctions.

The Holliday junction is a four-stranded DNA intermediate that arises during recombination reactions. We have designed and constructed a set of Holliday junction analogs that model each of the ideal conformations available to a 2-fold symmetric four-arm junction. The strategy used is to connect two arms of a junction molecule with a short tether of thymidines. These DNA molecules share a common core sequence but have different arms that are connected so that each molecule is constrained in either an antiparallel or a parallel structure. For tethered antiparallel molecules the identity of the crossover strands is determined by which arms are connected. Different arm connections gave molecules representing each of the two antiparallel crossover isomers. Two parallel molecules that differ in the length and position of the tether exhibit opposite biases in their choice of crossover strands. Thus, a physical constraint applied at a distance from the branch point can determine the conformation of a junction.

Base Sequence

Drug binding by branched DNA: selective interaction of the dye stains-all with an immobile junction.

The thiacarbocyanine dye Stains-All (4,5:4',5'-dibenzo-3,3'-diethyl-9-methylthiacarbocyanine bromide) is one of a large number of cyanine dyes introduced as photosensitizers in the photographic industry. Stains-All is used in histology as a stain for nucleic acids, proteins, polysaccharides, and lipids. We report here that the dye colors branched DNA molecules differently from linear duplexes and use footprinting experiments with methidiumpropyl-EDTA-Fe(II) [MPE.Fe(II)] and bis(o-phenanthroline)copper(I) [(O-P)2Cu(I)] to show that Stains-All interacts preferentially at the branch point of a four-arm DNA structure. A titration experiment allows us to estimate that the interaction of the dye with the branch has a dissociation constant below 45 nM, tighter than that of ethidium or methidium by over 2 orders of magnitude. Probing the interaction with the purine-specific reagent diethyl pyrocarbonate (DEPC) implies that the dye induces an asymmetric distortion near the branch in the major grooves of double helix in the junction.

Absorption

Drug binding by branched DNA: selective interaction of tetrapyridyl porphyrins with an immobile junction.

The differential binding of a number of water-soluble cationic porphyrins to a branched DNA molecule is reported. Tetrakis(4-N-methylpyridiniumyl)porphine (H2TMpyP-4) interacts near the branch point with an immobile DNA junction formed from four 16-mer strands. Its Cu(II) and Ni(II) derivatives show stronger preferential binding in the neighborhood of the branch point. Axially liganded derivatives, Zn, Co, and Mn, also interact near this branch point, but in a different way. We use the reagents methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and bis(o-phenanthroline)copper(I) [(OP)2Cu(I)] to cleave complexes of DNA duplex controls and the junction with these porphyrins. The resulting cleavage patterns are consistent with previous evidence that the branch point provides a strong site for intercalative binding agents, which is not available in unbranched duplexes of identical sequence. The preferential scission by (OP)2Cu(I) in the presence of Ni and Cu porphyrins near the branch point exceeds that seen for any agents we have studied. This hyperreactivity is not seen in the case of porphyrins with axial ligands, ZnTMpyP-4, CoTMpyP-4, and MnTMpyP-4, although these also interact near the branch point. The Zn derivative tends to protect sites close to the branch point from cutting, while the Co and Mn porphyrins moderately enhance cleavage of sites in this region.

Base Sequence

Drug binding by branched DNA molecules: analysis by chemical footprinting of intercalation into an immobile junction.

Branched DNA structures interact with drugs differently from unbranched control duplexes of similar sequence. A specific interaction between the reagent (methidiumpropyl-EDTA).Fe(II) [MPE.Fe(II)] and a branched DNA molecule formed from 16-mer oligonucleotide strands has been reported [Guo, Q., Seeman, N. C., & Kallenbach, N. R. (1989) Biochemistry 28, 2355-2359]. The structure of the branched molecule is thought to be made up of two double-helical stacking domains with an overall twofold symmetry across the branch site. The MPE-Fe(II) interaction occurs predominantly at or adjacent to the branch site and is eliminated by a second intercalator, propidium iodide. Further studies on the nature and properties of this site are presented here. Comparison of the patterns of scission of linear duplex and branched tetramer by EDTA.Fe(II), MPE.Fe(II), and Cu(I)-(o-phenanthroline)2 [(OP)2Cu(I)] provides a higher resolution picture of the site of enhanced binding. In particular, the sensitive footprinting afforded by (OP)2Cu(I) allows us to localize the major site of preferential interaction with propidium precisely to the branch point itself, with a roughly twofold symmetric pattern of cuts resulting. In detail, the differential pattern with respect to each duplex control is distinct for each arm of the junction. Excess propidium results in apparent reversal of the crossover isomer of the junction, indicating a possible additional avenue for the action of drugs in biological systems--effects on the products of recombination.

Base Sequence

De novo design of sequences for nucleic acid structural engineering.

An interactive procedure has been developed to assign sequences for the design of nucleic acid secondary structure. The primary goal of the procedure is to facilitate macromolecular architecture studies through the design of branched nucleic acid mono- and oligo-junction constructs in a convenient fashion. The essential feature of the sequence-symmetry minimization algorithm employed is the treatment of short sequences as vocabulary elements whose repetition decreases control over the resulting secondary structure. Both manual and semi-automatic application of this approach are available. The design of linear nucleic acid molecules or molecules containing single-stranded loops or connectors is also possible through application of the procedure.

Algorithms

Cruciform cutting endonucleases from Saccharomyces cerevisiae and phage T4 show conserved reactions with branched DNAs.

We have purified a cruciform DNA resolving endonuclease (Endo X3) greater than 1000-fold from crude extracts of mitotically growing Saccharomyces cerevisiae. The enzyme shows high specificity for DNAs with secondary structures and introduces characteristic patterns of staggered 'nicks' in the immediate vicinity of the structure. The following substrates were analyzed in detail: (i) naturally occurring four-way X junctions in cruciform DNA of a supercoiled plasmid; (ii) synthetic four-way X junctions with arms of 9 bp; (iii) synthetic three-way Y junctions with arms of 10 bp; and (iv) heteroduplex loops with 19 nucleotides in the loop. Cleavages were always found in the double stranded portion of the DNA, located immediately adjacent to the junction of the respective structure. The Endo X3 induced cleavage patterns are identical or very similar to the cleavage patterns induced in the same substrates by endonuclease VII (Endo VII) from phage T4. Furthermore, the activity of Endo X3 is completely inhibited in the presence of anti-Endo VII antiserum. Endo X3 has an apparent mol. wt of 43,000 daltons, determined by gel filtration and of approximately 18,000 daltons in SDS--polyacrylamide gels. Maximum activity of the enzyme was obtained in the presence of 10 mM MgCl2 at 31 degrees C in Tris-HCl buffer over a broad pH range with a maximum approximately 8.0. About 70% of maximal activity was obtained when Mg2+ was replaced by equimolar amounts of Mn2+ or Ca2+.

Base Sequence

DNase I cleavage of branched DNA molecules.

We report here a potentially useful signature of branched DNA structures. The base 5' to the branch and the five bases flanking the 3' side of the branch site are protected from cleavage by DNase I in both three- and four-arm branched DNA molecules. Our procedure is to measure the cleavage profile for each 5' -labeled strand in a control duplex and compare this with that of the same strand in a branched structure under conditions yielding less than one cut per strand. The resulting cleavage pattern in an immobile four-arm junction is roughly 2-fold symmetric, consistent with the pattern of Fe(II).EDTA-induced cleavage that has been observed previously. In the three-arm junction, the DNase I cleavage pattern is asymmetric, indicating lack of 3-fold symmetry. A variable pattern of protection occurs to the 5' side of the branch in some strands only for both three- and four-arm junctions, extending 2-4 residues 5' to the branch.

Base Sequence

Site-specific interaction of intercalating drugs with a branched DNA molecule.

The interaction of a stable branched DNA molecule with an intercalative drug is probed by hydroxyl radical scission. Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)], consisting of an intercalating ring system tethered to EDTA.Fe(II), produces the hydroxyl radicals by means of a Fenton reaction. The cleavage patterns of each labeled strand in a branched tetramer of four 16-mers are compared with those of the same strands in unbranched duplex controls. Strong differences between the profiles corresponding to scission of branched and duplex DNA molecules are seen in each of the strands at low MPE/DNA ratios. A specific site in the branched structure interacts preferentially with the drug, while other regions of the molecule are protected from cleavage. At 4 degrees C, cutting at strand positions demarcating the site of enhanced affinity is observed to be 60-100% more efficient than at the corresponding sequence positions in the control duplex DNA molecules; the degree of protection is comparable. Cleavage in the vicinity of the preferred site occurs at residues flanking the branch point. The reactive Fe(II) group appears to be centered within two residues of the branch point, and the site of preferential intercalation may be between the two base pairs abutting the branch point in one of the two helical domains. The pattern of preferential cutting at this site is eliminated in the presence of excess propidium diiodide, another intercalative drug.

DNA