Graham D. Hamblin

Rolling Circle Amplification-Templated DNA Nanotubes Show Increased Stability and Cell Penetration Ability

DNA nanotubes hold promise as scaffolds for protein organization, as templates of nanowires and photonic systems, and as drug delivery vehicles. We present a new DNA-economic strategy for the construction of DNA nanotubes with a backbone produced by rolling circle amplification (RCA), which results in increased stability and templated length. These nanotubes are more resistant to nuclease degradation, capable of entering human cervical cancer (HeLa) cells with significantly increased uptake over double-stranded DNA, and are amenable to encapsulation and release behavior. As such, they represent a potentially unique platform for the development of cell probes, drug delivery, and imaging tools.

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.

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.

Simple design for DNA nanotubes from a minimal set of unmodified strands: rapid, room-temperature assembly and readily tunable structure.

DNA nanotubes have great potential as nanoscale scaffolds for the organization of materials and the templation of nanowires and as drug delivery vehicles. Current methods for making DNA nanotubes either rely on a tile-based step-growth polymerization mechanism or use a large number of component strands and long annealing times. Step-growth polymerization gives little control over length, is sensitive to stoichiometry, and is slow to generate long products. Here, we present a design strategy for DNA nanotubes that uses an alternative, more controlled growth mechanism, while using just five unmodified component strands and a long enzymatically produced backbone. These tubes form rapidly at room temperature and have numerous, orthogonal sites available for the programmable incorporation of arrays of cargo along their length. As a proof-of-concept, cyanine dyes were organized into two distinct patterns by inclusion into these DNA nanotubes.

Sequence-responsive unzipping DNA cubes with tunable cellular uptake profiles

Here, we demonstrate a new approach for the design and assembly of a dynamic DNA cube with an addressable cellular uptake profile. This cube can be selectively unzipped from a 3D to a flat two-dimensional structure in the presence of a specific nucleic acid sequence. Selective opening is demonstrated in vitro using a synthetic RNA marker unique to the LNCaP human prostate cancer cell line. A robust uptake in LNCaP cells, HeLa cells (human cervical cancer) and primary B-lymphocytes isolated from the blood of chronic lymphocytic leukemia (CLL) patients is observed using fluorescence-activated cell sorting (FACS), confocal microscopy and a new cluster analysis algorithm combined with image cross-correlation spectroscopy. The DNA cube was modified with hydrophobic and hydrophilic dendritic chains that were found to coat its exterior. The dynamic unzipping properties of these modified cubes were retained, and assessment of cellular uptake shows that the hydrophobic chains help with the rapid uptake of the constructs while the hydrophilic chains become advantageous for long term internalization.

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.

Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis

Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNAMet in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.

Sequential growth of long DNA strands with user-defined patterns for nanostructures and scaffolds

DNA strands of well-defined sequence are valuable in synthetic biology and nanostructure assembly. Drawing inspiration from solid-phase synthesis, here we describe a DNA assembly method that uses time, or order of addition, as a parameter to define structural complexity. DNA building blocks are sequentially added with in-situ ligation, then enzymatic enrichment and isolation. This yields a monodisperse, single-stranded long product (for example, 1,000 bases) with user-defined length and sequence pattern. The building blocks can be repeated with different order of addition, giving different DNA patterns. We organize DNA nanostructures and quantum dots using these backbones. Generally, only a small portion of a DNA structure needs to be addressable, while the rest is purely structural. Scaffolds with specifically placed unique sites in a repeating motif greatly minimize the number of components used, while maintaining addressability. This combination of symmetry and site-specific asymmetry within a DNA strand is easily accomplished with our method.

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.

Stoichiometry and Dispersity of DNA Nanostructures Using Photobleaching Pair-Correlation Analysis

A wide variety of approaches have become available for the fabrication of nanomaterials with increasing degrees of complexity, precision, and speed while minimizing cost. Their quantitative characterization, however, remains a challenge. Analytical methods to better inspect and validate the structure and composition of large nanoscale objects are required to optimize their applications in diverse technologies. Here, we describe single-molecule fluorescence-based strategies relying on photobleaching and multiple-color co-localization features toward the characterization of supramolecular structures. By optimizing imaging conditions, including surface passivation, excitation power, frame capture rate, fluorophore choice, buffer media, and antifading agents, we have built a robust method by which to dissect the structure of synthetic nanoscale systems. We showcase the use of our methods by retrieving key structural parameters of four DNA nanotube systems differing in their preparation strategy. Our method rapidly and accurately assesses the outcome of synthetic work building nano- and mesoscale architectures, providing a key tool for product studies in nanomaterial synthesis.