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.

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 tRNA(Met) 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.

Gold nanoparticle 3D-DNA building blocks: high purity preparation and use for modular access to nanoparticle assemblies.

Using highly functional 'building-blocks' of AuNPs mono-conjugated to three-dimensional DNA 'rung' structures, both discrete and extended linear assemblies are controllably prepared via addition of various templating backbone strands. This unique approach presents a facile alternative to other methods of AuNP organization through DNA, and has potential utility in the fields of nanophotonics and nanoelectronics.

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.

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.

Supramolecular DNA assembly.

The powerful self-assembly features of DNA make it a unique template to finely organize and control matter on the nanometre scale. While DNA alone offers a high degree of fidelity in its self-assembly, a new area of research termed 'supramolecular DNA assembly' has recently emerged. This field combines DNA building blocks with synthetic organic, inorganic and polymeric structures. It thus brings together the toolbox of supramolecular chemistry with the predictable and programmable nature of DNA. The result of this molecular partnership is a variety of hybrid architectures, that expand DNA assembly beyond the boundaries of Watson-Crick base pairing into new structural and functional properties. In this tutorial review we outline this emerging field of study, and describe recent research aiming to synergistically combine the properties inherent to DNA with those of a number of supramolecular scaffolds. This ultimately creates structures with numerous potential applications in materials science, catalysis and medicine.

Cell-selective proteomics for biological discovery.

Cells alter the proteome to respond to environmental and developmental cues. Global analysis of proteomic responses is of limited value in heterogeneous environments, where there is no 'average' cell. Advances in sequencing, protein labeling, mass spectrometry, and data analysis have fueled recent progress in the investigation of specific subpopulations of cells in complex systems. Here we highlight recently developed chemical tools that enable cell-selective proteomic analysis of complex biological systems, from bacterial pathogens to whole animals.

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.

Stepwise growth of surface-grafted DNA nanotubes visualized at the single-molecule level.

DNA nanotubes offer a high aspect ratio and rigidity, attractive attributes for the controlled assembly of hierarchically complex linear arrays. It is highly desirable to control the positioning of rungs along the backbone of the nanotubes, minimize the polydispersity in their manufacture and reduce the building costs. We report here a solid-phase synthesis methodology in which, through a cyclic scheme starting from a 'foundation rung' specifically bound to the surface, distinct rungs can be incorporated in a predetermined manner. Each rung is orthogonally addressable. Using fluorescently tagged rungs, single-molecule fluorescence studies demonstrated the robustness and structural fidelity of the constructs and confirmed the incorporation of the rungs in quantitative yield (>95%) at each step of the cycle. Prototype structures that consisted of up to 20 repeat units, about 450 nm in contour length, were constructed. Combined, the solid-phase synthesis strategy described and its visualization through single-molecule spectroscopy show good promise for the production of custom-made DNA nanotubes.

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.

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.

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.

Loading and selective release of cargo in DNA nanotubes with longitudinal variation.

Nanotubes hold promise for a number of biological and materials applications because of their high aspect ratio and encapsulation potential. A particularly attractive goal is to access nanotubes that exert well-defined control over their cargo, such as selective encapsulation, precise positioning of the guests along the nanotube length and triggered release of this cargo in response to specific external stimuli. Here, we report the construction of DNA nanotubes with longitudinal variation and alternating larger and smaller capsules along the tube length. Size-selective encapsulation of gold nanoparticles into the large capsules of these tubes leads to 'nanopeapod' particle lines with positioning of the particles 65 nm apart. These nanotubes can then be opened when specific DNA strands are added to release their particle cargo spontaneously. This approach could lead to new applications of self-assembled nanotubes, such as in the precise organization of one-dimensional nanomaterials, gene-triggered selective delivery of drugs and biological sensing.

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.

A theoretical study of Favorskii reaction stereochemistry. Lessons in torquoselectivity.

The mechanisms of the chloroenolate-->cyclopropanone step of the "normal" Favorskii rearrangement have been investigated in detail using high-level ab initio calculations. A series of simple alpha-chloroenolates, based on chloroacetone (6), all monomethyl derivatives (7-9), a dimethyl analogue (10), and 1-acetyl-1-chlorocyclohexane (11) was first used to explore and define the basic features of the mechanism, which include the finding of both an "inversion" and a "retention" transition state and that in most cases these arise from separate ground-state conformations of the chloroenolate. These theoretical studies were then extended to an isomeric pair of chloroenolates 1 and 2, the cis- and trans-2-methyl derivatives of 11, which are the reactive intermediates involved in a well-known experimental study carried out by Stork and Borowitz (S-B). Finally, three alpha-chlorocyclohexanone enolate systems 12-14 were studied, since these intermediates have a more restricted enolate geometry. The "inversion" mechanism has been described as an SN2 process but the present results, while supporting a concerted process, is better described as an oxyallyl structure undergoing concerted ring closure. The "retention" mechanism has been described as SN1-like, but the calculations show that this process is also concerted, although much less so, and again involves oxyallyl-like transition-states. The model systems 6-8, 10, and 11 with a potential plane of symmetry have two enantiomeric transition states for inversion and another two for retention of configuration (at the C-Cl center). With 9 and the S-B models 1 and 2, with no symmetry plane, there are a calculated total of four diastereomeric transition states for cyclopropanone ring closure in each case, two for inversion and two for retention. While the transition-state energies calculated for simple chloroenolates favor the inversion process, the S-B models 1 and 2 have almost equal inversion-retention transition-state energies. Solvation simulation calculations of ground states and transition states suggest that the retention mechanism becomes relatively more favored in polar solvents, in agreement with some experimental results. In the chloroenolates 12-14, both inversion and retention mechanisms were also located, these arising from two different ground-state ring conformations of the enolate. In these models, one also finds similar inversion and retention transition-state energies, but again with a small preference for the inversion process.