Programable self-assembly of three-dimensionally defined nano objects
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Programable self-assembly of three-dimensionally defined nano objects

DNA Nanoconstruction, Scaffolds and Cages
DNA nanoconstruction benefits from the structural rigidity of short dsDNA, scalability, good accessibility of synthetic and chemically modified DNA and the option of enzymatic amplification and processing.


Fig. 1.2.1 DNA nanoconstruction:

a) Molecular model of the DNA cube (top) and the truncated octahedron (bottom).
b) Design of the DNA octahedron (left), 3D model derived from cryo EM data (right), and secondary structure of the branched-tree folding intermediate (bottom); colours indicate half paranemic crossover loops, coloured stripes coincide with strand crossover positions. Folding to the structure in the upper left is complete when all seven paranemic cross-over struts have formed
c) Design of a DNA tetrahedron formed by annealing four oligonucleotides (1-4). Complemen-tary subsequences that hybridize to form the edges are identified by color.
d) A DNA tetrahedron formed by the self-assembly of four trisoligonucleotides. Colors indicate complementary sequences.

A number of groups have succeeded in building a variety of two- and three-dimensional structures out of DNA oligomers. To assemble objects with the linear DNA molecule, branching elements have to be employed. Seeman´s early proposal from 1982 (i) suggested branched DNA motifs based on the Holliday junction, a junction between four DNA strands that is known as an intermediate from DNA recombination in vivo. Overhanging single-stranded DNA at the termini of each arm, so-called sticky ends, allow the programmable self-assembly of DNA junctions with complementary overhangs. DNA junctions with three (ii), four, five or six (iii), eight and twelve (iv) arms have been realized so far. Further bio-inspired DNA branching elements such as double and triple crossover motifs (two or three helices interconnected by strand exchange) were developed to achieve more rigid branching elements for DNA nanoconstruction (v). Furthermore, the usage of paranemic crossover motifs allows interconnections of topologically closed DNA structures, analogous to loop-loop interactions known from RNA (vi). Alternatively, branched DNA can be obtained using chemically modified nucleotides or artificial organic linker molecules to connect three or more oligonucleotides covalently. The first three-dimensional DNA object was reported by Seeman and coworkers in 1991. The object possessed the topology of a cube and was generated by self-assembly of DNA junctions with complementary sticky ends. In a stepwise process a construct of six adequately interlocked DNA rings was synthesized. In each step a new face was generated by hybridization of DNA junctions followed by enzymatic ligation and purification. The edges of the resulting DNA polyhedra consisted of dsDNA; the DNA junctions represented the vertices. After refinement of the synthesis protocol Seeman´s lab also reported the synthesis of a truncated octahedron (vii). However, the yields of the laborious syntheses were low (in the order of 0.1 %) and the resulting DNA polyhedra presumably lack rigidity since the employed DNA junctions are flexible constructs (Fig. 1.2.1a).

Since then a much wider range of 3-D objects have been construct, many with specific capabilities. Shih et al. synthesized a “clonable” DNA octahedron using rigid double crossover and paranemic crossover motifs (viii). A cloneable polynucleotide (1669 nucleotides) and five shorter oligonucleotides (40 nucleotides) were designed to create the octahedral object in a hierarchical self-assembly process. The self-assembly of the DNA strands led to an initial branched-tree folding intermediate structure, which was composed of twelve struts (octahedron edges) connected by six four-way junctions (octahedron vertices). Five of the octahedron edges were represented by double crossover motifs while the remaining seven edges were formed by paranemic crossover motifs during a second self-assembly step. Examination of the self-assembly product by cryogenic electron microscopy (cryo-EM) revealed an object with octahedral shape (Fig. 1.2.1b). More recently the synthesis of a covalently closed octahedral DNA object was reported by the group of Knudsenix. Mao and coworkers synthesized DNA tetrahedra, dodecahedra and “buckyballs” in a hierarchical self-assembly process (x). A three-point-star motif with sticky ends was generated from three different types of oligonucleotides. Depending on concentration and flexibility of the three-point-star motif, tetrahedra, dodecahedra or buckyballs resulted from the self-assembly. When using a five-point-star motif with fine-tuned conformational flexibility, the authors observed the formation of icosahedral DNA objects (xi). Rigid DNA cubes were generated by the self-assembly of two types of suitably “oriented” three-point-star motifs (xii). Very recently, the group of Krishnan reported the generation of icosahedral objects, which were step-wise assembled from a set of sticky-ended DNA five-way junctions (xiii). Each “hemisphere” of the icosahedron was synthesized from a set of six of five-way junctions: A central five-way junction hybridized with another five five-way junctions and was subsequently chemically ligated. The two “hemispheres” were brought together in a 1:1 ratio and assembled into the icosahedral structure, which could be chemically ligated to enhance the stability of the object. The authors showed in electron micrographs that gold nanoparticles could be encapsulated in the interior of the DNA icosahedron. Interestingly, a depletion of small gold nanoparticles in case of the encapsulated nanoparticles was observed, indicating that small nanoparticles can pass the meshes of the DNA capsule. The group of Turberfield reported on the synthesis of tetrahedral DNA nanostructures from a single-step self-assembly of four linear oligonucleotides (Fig. 1.2.1c) (xiv). Furthermore, the authors were able to show that a nano-sized object (cytochrome c protein), covalently fixed at a nucleobase of one oligonucleotide, could be positioned inside or outside the nano-cage (xv). Following the same strategy but using six oligonucleotides instead of four also a trigonal bipyramid was generated in a one-step self-assembly(xvi). Decoration of objects with various compounds has been achieved as reported by Distefano (xvii) and Mitchell (xviii) which opened up the door for tethering of the polyhedra.
Additional opportunities arise from covalently branched DNA in which modified nucleotides or artificial linkers connect three or more oligonucleotides. (xix,xx,xxi,xxii,xxiii,xxiv,xxv,xxvi,xxvii,xxviii,xxix,xxx,xxxi,xxxii,xxxiii) Some branching monomers for the synthesis of symmetric and asymmetric branched oligonucleotides have been commercially available for several years(xxxiv). It must be stressed however, that the utilization of these materials yield diasteromeric mixtures of branched DNA, which in turn give mixtures of 2N different 3D-objects having N vertices. So called trisoligonucleotides were developed to overcome such complication. The simplest class consists of three identical linear oligonucleotides that are 3‟-connected by an organic linker molecule (xxxiv). Depending on the annealing protocol a set of two complementary trisoligonucleotides self-assembled either into supramolecular networks (slow cooling of the mixture) or into discrete objects such as dimers, trimers, tetramers etc. (fast cooling, followed by heating to annealing temperature). This finding was interpreted as thermodynamically versus kinetically controlled self-assembly. Dorenbeck synthesized a set of four trisoligonucleotides with individual sequences, designed to encode the connectivity of a tetrahedron (Fig. 1.2.1d)(xxxv). Eckardt et al. did a first attempt towards replicable trisoligonucleotide-based nanoconstruction as they developed a process that enables the copying of the connectivity information of trisoligonucleotides (xxxvi). More importantly larger structures have recently been reported by Zimmermann et al. (xxxvii) who developed a third generation of trisoligonucleotides based on C3h-symmetric linker elements. When uniformly connected at their 3‟-end all arms are programmed to experience identical conformational constraints by the linker. This in turn enabled a successful one-step self-assembly of a dodecahedral object in high yield. The set of 20 trisoligos contained 60 sequences which were designed for similar Tm using Banzhaf‟s DNA sequence generator (xxxviii). Furthermore, the addressable functionalization of the DNA object was realized by the introduction of overhang strands which could be functionalized at defined position by hybridization with a complementary strand modified with a fluorescent dye or a gold nanoparticle (xxxvii). Very recently, a theoretical concept for the self-assembly of DNA-cages proposed the usage of DNA grafted nanoparticles (xxxix). Our approach is different as it will select for the proper inclusion and do the informationally controlled grafting inside. When comparing C3h-trisoligonucleotide-based 3D-nanoconstruction with other approaches we see the following advantages:

  • Trisoligonucleotides are artificial building blocks whose properties can be tuned by the nature of the linker molecule.
  • Trisoligonucleotide nanoconstruction is vertex-based; viz. keeping the modular advantages of a vertex-based approach and combining it with the high yields of noncovalent self-assembly.
  • The artificial linker replaces an informational load of about 60-120 nucleotides otherwise needed to encode the central part of a functionally equivalent noncovalent 3-way junction.
  • Protocols are available that allow the synthesis of all possible types of trisoligonucleotides: 3x 3‟-linked, 3x 5‟-linked, 2x 3‟- and 1x 5‟-linked, and 1x 3‟- and 2x 5‟-linked.
  • The arms may either have identical sequences or up to 3 different sequences. 1, 2, or 3 arms may contain sequence information beyond what is needed to encode the connectivity of the polyhedral object.
  • Trisoligo synthesis employs palladium-based protection group chemistry, which is non-standard for most DNA synthesizers today. Nevertheless, the yields are only slightly below those of linear trisoligonucleotides of the same number of nucleotides. With the latest synthesizer equipment we expect to be able also to automate the non-standard steps. Then the total synthetic effort and costs are maximally one third and realistically one tenth of the approach using noncovalent 3-way junctions.
  • Trisoligonucleotide objects combine the highest informational capacity for multi-modular scaffolding with the smallest possible size of the scaffolds. Turberfield‟s edge/face-based approach should in principle enable one to attach as many modules as there are faces (e.g. 12 for a dodecahedron). Using our strategy employing trisoligonucleotide extension strands this number is expected to be 60, viz. 5 times higher.
  • Mao‟s strategy, namely to rigidify noncovalent Seeman-type junctions and to control the angle and orientation of the arms in space is a perfect one if the goal is to arrive at large polyhedral objects which are nicely visible in Cryo-EM (like in the case of Shi‟s octahedron). However the strategy is NOT based on connectivity information. It could be expanded into this direction, but then the synthetic efforts (determining the cost for a single “utility kit”) are expected to be 2 orders of magnitude higher than in the case of trisoligo-based kits.
  • Trisoligonucleotide assemblies are not necessarily rigid by themselves. Tetrahedra are, but dodecahedra, cubes, etc. are not due to a lack of tensegrity. For MATCHIT however we see this issue as an advantage: The ribosome - being THE natural prototype of an information-processing nanomachine – is not rigid due its scaffold rRNA. The scaffold however encodes the positional assembly of 51 proteins, from which the integrity of the ribozyme emerges.
  • It should be noticed that the dodecahedral scaffolding is a “Poor Man‟s” approach to nano-scaffolding and we do not see any clear advantage to use a much more expensive origami box scaffoldings. Further, we do not know which potential additional compatibility issues we may have using the origami boxes in connection to the micro containers.

The vision behind these issues is to arrive at a chemical implementation of a prototype generation of utility foglets, viz. hypothetical nanoscale robots that can grab and release chemical modules at defined and programmable positions, orchestrated positional synthesis and catalysis and also self-assembly into larger aggregates. This vision however accepts chemistry as a science and does not seek for a “diamondoid” replacement of chemistry. State of the art and beyond is illustrated in Figure 1.2.2.


Fig. 1.2.2 State of the art and beyond:
a) Molecular model of a DNA dodecahedron, self-assembled from 20 trisoligonucleotides.
b) AFM image of dodecahedra adsorbed on mica.
c) Cryo-EM class averages (top row) and 3D processed (bottom row) images of the naked dodecahedra.
d) A utility foglet (left) self-assembles into a 4 x 5 grid (right).

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