Christopher K. McLaughlin

Metal-nucleic acid cages.

Metal-nucleic acid cages are a promising new class of materials. Like metallo-supramolecular cages, these systems can use their metals for redox, photochemical, magnetic and catalytic control over encapsulated cargo. However, using DNA provides the potential to program pore size, geometry, chemistry and addressability, and the ability to symmetrically and asymmetrically position transition metals within the three-dimensional framework. Here we report the quantitative construction of metal-DNA cages, with the site-specific incorporation of a range of metals within a three-dimensional DNA architecture. Oligonucleotide strands containing specific environments suitable for transition-metal coordination were first organized into two DNA triangles. These triangles were then assembled into a DNA prism with linking strands. Metal centres were subsequently incorporated into the prisms at the pre-programmed locations. This unprecedented ability to position transition metals within a three-dimensional framework could lead to metal-DNA hosts with applications for the encapsulation, sensing, modification and release of biomolecules and nanomaterials.

Modular construction of DNA nanotubes of tunable geometry and single- or double-stranded character

DNA nanotubes can template the growth of nanowires, orient transmembrane proteins for nuclear magnetic resonance determination, and can potentially act as stiff interconnects, tracks for molecular motors and nanoscale drug carriers. Current methods for the construction of DNA nanotubes result in symmetrical and cylindrical assemblies that are entirely double-stranded. Here, we report a modular approach to DNA nanotube synthesis that provides access to geometrically well-defined triangular and square-shaped DNA nanotubes. We also construct the first nanotube assemblies that can exist in double- and single-stranded forms with significantly different stiffness. This approach allows for parameters such as geometry, stiffness, and single- or double-stranded character to be fine-tuned, and could enable the creation of designer nanotubes for a range of applications, including the growth of nanowires of controlled shape, the loading and release of cargo, and the real-time modulation of stiffness and persistence length within DNA interconnects.

Three-dimensional organization of block copolymers on "DNA-minimal" scaffolds.

Here, we introduce a 3D-DNA construction method that assembles a minimum number of DNA strands in quantitative yield, to give a scaffold with a large number of single-stranded arms. This DNA frame is used as a core structure to organize other functional materials in 3D as the shell. We use the ring-opening metathesis polymerization (ROMP) to generate block copolymers that are covalently attached to DNA strands. Site-specific hybridization of these DNA-polymer chains on the single-stranded arms of the 3D-DNA scaffold gives efficient access to DNA-block copolymer cages. These biohybrid cages possess polymer chains that are programmably positioned in three dimensions on a DNA core and display increased nuclease resistance as compared to unfunctionalized DNA cages.

Templated Ligand Environments for the Selective Incorporation of Different Metals into DNA

DNA has emerged as a unique template for the construction and organization of nanostructures and arrays with precisely controlled features. The incorporation of transition metals into DNA has enabled the transfer of functionality, in the form of enhanced stability, redox activity, photoactivity, and magnetic and catalytic properties, to this otherwise passive biomolecular template. A particularly attractive goal would be the selective incorporation of different transition metals into DNA. This allows the use of the programmable character of DNA to organize transition metals into arbitrarily designed symmetric or asymmetric structures, resulting in a number of applications in artificial photosynthesis, multimetallic catalysis, nanooptics, nanoelectronics, and data storage. It would also result in the expansion of the DNA “alphabet” to new metal “letters” that would increase the information content of this biomolecule and reduce errors in its assembly into nanostructures. For this goal to reached, different DNA–ligand environments must be designed in a manner that maximizes metalbinding selectivity, promotes close interaction between the metal complex and the DNA duplex, and offers coordination programmability. The incorporation of metals into DNA constructs has been demonstrated through replacement of the hydrogen-bonded DNA base pairs with metal complexes within the interior of the DNA duplex. 3] The extension of this strategy to different ligand environments is, however, limited by the steric and spatial requirements of the DNA duplex, and has been successful for planar metal centers that fit in the DNA interior. Metal complexes have been appended as nucleobase and (deoxy)ribose modifications; however, this method is generally limited to a small subset of kinetically inert and unreactive metal complexes that resist the harsh conditions of automated DNA synthesis. A third approach developed by our research group is to insert ligands into the phosphodiester backbone, such that the hybridization of DNA with its complementary strand templates the assembly of a metal-coordination environment in close contact with the DNA base stack. We report herein the site-specific incorporation of terpyridine (tpy) and diphenylphenanthroline (dpp) ligands into DNA strands. The DNAtemplated creation of three ligand environments resulted: tpy2:DNA, tpy:dpp:DNA, and dpp2:DNA. These ligand environments are highly selective for six-, five-, and fourcoordinate metal ions, respectively (Figure 1a). Thermal denaturation, UV/Vis and circular dichroism spectroscopy, and gel electrophoresis revealed a strong preference of the octahedral metal ions Fe and Co for the tpy2:DNA environment, with the highest reported thermal denaturation increase of any DNA structure upon Fe coordination. The dpp2:DNA environment is highly selective for the tetrahedral Cu ion, whereas tpy:dpp:DNA exhibits preference for the five-coordinate Cu ion. Moreover, “error correction” was observed if a metal ion was placed in the incorrect environment. Thus, Cu spontaneously oxidized to Cu if added to tpy:dpp:DNA, and Cu underwent spontaneous reduction to Cu if placed in dpp2:DNA. The four-coordinate Ag I ion was displaced by Cu when placed in the tpy:dpp:DNA structure. Finally, the addition of Fe to the tpy:dpp:DNA structure resulted in reorganization of the ligand environment, such that two of these constructs were brought together with Fe binding to their terpyridine units. In a similar manner to the ligand pockets of metalloenzymes, this new class of DNAtemplated coordination environments defines a toolbox for the selective positioning of different transition metals at exact locations within DNA nanostructures. Details of the synthesis, characterization, and solid-phase incorporation of the phosphoramidite derivatives of the dpp and tpy ligands into the DNA (Figure 1c) can be found in the Supporting information. The tpy and dpp derivatives were both designed to have flexible diethylene glycol spacers that would enable placement of their aromatic moieties in close proximity to the DNA base stack to promote strong interaction and chirality transfer from the B-DNA duplex upon metal binding (Figure 1b). The selective insertion of tpy and dpp at the 5’ and 3’ termini of complementary DNA strands a and b, followed by hybridization, resulted in the formation of three DNA-templated coordination spheres: tpy2:ab, tpy:dpp:ab, and dpp2:ab (Figure 1a). In contrast to metal-driven supramolecular coordination, 10] the programmed self-assembly of duplex DNA can template the formation of any combination of ligands, whether homoleptic or mixed, in close proximity. We studied the thermal stability of DNA duplexes tpy2:ab and their metal complexes (Figure 2a). For these duplexes, even without the addition of a metal, the melting temperature increased from 43 8C for the unmodified DNA to 66 8C for tpy2:ab. This increase is possibly a result of p stacking of the tpy ligands on the DNA base stack, or interactions of tpy ligands with Na or H ions in the buffer solution. [*] H. Yang, A. Z. Rys, C. K. McLaughlin, Prof. H. F. Sleiman Department of Chemistry, McGill University 801 Sherbrooke Street West, Montreal, Quebec H3A 2K6 (Canada) Fax: (+ 1)514-398-3937 E-mail: hanadi.sleiman@mcgill.ca

Development and characterization of gene silencing DNA cages.

RNA interference (RNAi) is a powerful therapeutic strategy that induces gene silencing by targeting disease-causing mRNA and can lead to their removal through degradation pathways. The potential of RNAi is especially relevant in cancer therapy, as it can be designed to regulate the expression of genes involved in all stages of tumor development (initiation, growth, and metastasis). We have generated gene silencing 3D DNA prisms that integrate antisense oligonucleotide therapeutics at 1, 2, 4, and 6 positions. Synthesis of these structures is readily achieved and leads to the assembly of highly monodisperse and well-characterized structures. We have shown that antisense strands scaffolded on DNA cages can readily induce gene silencing in mammalian cells and maintain gene knockdown levels more effectively than single and double stranded controls through increased stability of bound antisense units.

Chiral Metal–DNA Four‐Arm Junctions and Metalated Nanotubular Structures

DNA is a promising template for the programmable assembly of nanostructures. A number of construction strategies have been developed, such as the weaving together of many DNA strands into “tiles” or the stapling of a long DNA strand into “origami” assemblies. These approaches use DNA as the only information source for organization and result in DNAdense, large, and rigid nanostructures. An interesting alternative is the introduction of synthetic molecules to create DNA assemblies with new structures and functions. The use of organic or inorganic molecules as junctions can eliminate the need to interweave DNA strands for structural definition and thus results in “DNA-economical” structures with smaller sizes and increased dynamic character. Transition-metal junctions are especially useful, because they can impart functional advantages, such as photophysical, redox, catalytic, and magnetic properties, as well as enhanced stability to DNA nanostructures. 6] Because of their diverse coordination geometries, transition metals may also have key structural advantages for DNA nanoconstruction. They can lead to junction architectures that are inaccessible with DNA alone and may be able to mediate the transfer of chirality from the DNA double helix to the junction itself. Such chirality transfer is particularly desirable, as many DNA junctions are intrinsically chiral because of sequence and groove asymmetry, and their assembly can lead to diastereomeric mixtures. Control of the chirality of these junctions can result in stereospecific, higher-yielding DNA-nanostructure syntheses and more specific interactions with other biological molecules. Herein we describe a DNA-templated method for the formation of a chiral metal–DNA junction containing a single copper(I)–bisphenanthroline unit at its central point and four single-stranded DNA arms of different sequences. This structure is the simplest, most compact four-arm junction derived from DNA and would be difficult to access without the mediation of the transition metal. The design, evolution, and optimization of the metal–ligand complex for efficient chirality transfer from DNA is reported. From this structure, we then constructed metal–DNA triangular rungs and formed the first metal–DNA nanotubular structures. In our approach to the construction of chiral junctions with four different arms, we use two DNA strands, D1 and D2, each modified in the middle of the 20-base sequence with a diphenylphenanthroline (dpp) ligand (Figure 1a). A template

A facile, modular and high yield method to assemble three-dimensional DNA structures.

We describe a rapid and quantitative method to generate DNA cages of deliberately designed geometry from readily available starting strands. Balancing the incorporation of sequence uniqueness and symmetry in a face-centered approach to 3D construction can result in triangular (TP), rectangular (RP), and pentagonal prisms (PP) without compromising the potential for nanostructure addressability.

Self-Assembly of Metal-DNA Triangles and DNA Nanotubes with Synthetic Junctions

The site-specific insertion of organic and inorganic molecules into DNA nanostructures can provide unique structural and functional capabilities. We have demonstrated the inclusion of two types of molecules. The first is a diphenylphenanthroline (dpp, 1) molecule that is site specifically inserted into DNA strands and which can be used as a template to create metal-coordinating pockets. These building blocks can then be used to assemble metal-DNA 2D and 3D structures, including metal-DNA triangles, described here. The second insertion is a triaryl molecule that provides geometric control in the preparation of 2D single-stranded DNA templates. These can be designed to further assemble into geometrically well-defined nanotubes. Here, we detail the steps involved in the construction of metal-DNA triangles and DNA nanotubes using these methods.

136 Dendritic Alkyl Chains on DNA Cages: A Geometry-Dependent Inter- or Intramolecular “Handshake”

The selective association of hydrophobic sidechains is a strong determinant of protein organization. We have observed a parallel mode of assembly in DNA nanotechnology. Firstly, dendritic DNA amphiphiles (D-DNA) were synthesized (Carneiro, Aldaye, & Sleiman, 2009) comprising an addressable oligonucleotide portion and a hydrophobic alkyl dendron at the 5’ terminus. DNA amphiphiles have gathered interest recently as they can self-assemble in aqueous media to form well defined micelles while also retaining the ability to hybridize to their complement (Kwak & Herrmann, 2011; Patwa, et al. 2011) Two variations of alkyl D-DNA were hybridized to the single-stranded edges of a DNA cube (McLaughlin, et al., 2012). It was found that anisotropic organization of these hydrophobic domains on the 3D scaffold results in a new set of assembly rules, dependent on spatial orientation, number, and chemical identity of the D-DNA on the cubic structure (Edwardson. et al. 2012). When four amphiphiles are organized on one cube face, the hydrophobic residues engage in an intermolecular “handshake” between two cubes, resulting in a dimer. When eight amphiphiles are organized on the top and bottom faces of the cube, they engage in a “handshake” inside the cube. Combining the highly specific recognition of the oligonucleotide sequence with the orthogonal association of hydrophobic moieties can lead to a variety of structures with such diverse applications as membrane anchoring, cell uptake, directed hydrophobic assembly, and encapsulation and release of small molecules.

137 DNA nanostructure serum stability: greater than the sum of their parts

DNA cages hold tremendous potential to encapsulate and selectively release therapeutic drugs, and can provide useful tools to probe the size and shape dependence of nucleic acid delivery (McLaughlin & Sleiman, H. F., 2011). These structures have been shown to site-specifically present ligands, small molecule drugs, or antisense/siRNA motifs, in order to increase their therapeutic efficiency (Li & Fan, C. 2012). One of the major barriers towards their in vivo applications is the susceptibility of their strands towards nuclease degradation. A number of chemical strategies have been used to block nuclease digestion of oligonucleotides and improve potency, such as the use of a phosphorothioate backbone, 2´-O-methyl, locked nucleic acids, and short hybrid gapmers. However, the synthesis of these oligonucleotides is often complicated and expensive, driving the need for simple modifications to enhance serum stability and address in vivo biodistribution. We show here a simple method to significantly enhance the nuclease stability of DNA strands, through introduction of commercially available, single-endmodifications (Conway & Sleiman 2013). We use these oligonucleotides to construct DNA cages in a single step and in quantitative yields. Even in single-stranded form, these cages stabilize their component strands towards nucleases, with mean lifetimes as long as 62 h in 10 % (v/v) fetal bovine serum (FBS). We examine the effect of other DNA-end modifications on nuclease susceptibility. Finally, we show the ligation of these single-stranded cages into topologically interesting catenane ‘necklaces,’ with mean lifetimes in serum of ∼200 h.