Synthesis and characterization of self-assembled DNA nanostructures.

The past decade witnessed the fast evolvement of structural DNA nanotechnology, which uses DNA as blueprint and building material to construct artificial nanostructures. Using branched DNA as the main building block (also known as a "tile") and cohesive single-stranded DNA (ssDNA) ends to designate the pairing strategy for tile-tile recognition, one can rationally design and assemble complicated nanoarchitectures from specifically designed DNA oligonucleotides. Objects in both two- and three-dimensions with a large variety of geometries and topologies have been built from DNA with excellent yield; this development enables the construction of DNA-based nanodevices and DNA-template directed organization of other molecular species. The construction of such nanoscale objects constitutes the basis of DNA nanotechnology. This chapter describes the protocol for the preparation of ssDNA as starting material, the self-assembly of DNA nanostructures, and some of the most commonly used methods to characterize the self-assembled DNA nanostructures.

DNA self-assembly for nanomedicine.

Self-assembling DNA nanostructures based on rationally designed DNA branch junction molecules has recently led to the construction of patterned supramolecular structures with increased complexities. An intrinsic value of DNA tiles and patterns lies in their utility as molecular pegboard for deterministic positioning of molecules or particles with accurate distance and architectural control. This review will discuss the state-of-art developments in self-assembled DNA nanostructural system. Biomedical aspects of information guided DNA nanostructures will also be summarized. We illustrate both the use of simple DNA artworks for sensing, computation, drug delivery and the application of more complex DNA architectures as scaffolds for the construction of protein and nanoparticle arrays.

One-pot nucleation, growth, morphogenesis, and passivation of 1.4 nm Au nanoparticles on self-assembled rosette nanotubes.

A one-pot strategy for the nucleation, growth, morphogenesis, and passivation of 1.4 nm Au nanoparticles (NPs) on self-assembled rosette nanotubes (RNTs) is described. Tapping-mode atomic force microscopy, transmission electron microscopy, energy-dispersive X-ray analysis, and selected-area electron diffraction were used to establish the structure and organization of this hybrid material. Notably, we found that the Au NPs formed were nearly monodisperse clusters of Au(55) (1.4-1.5 nm) nestled in pockets on the RNT surface.

Distance-dependent interactions between gold nanoparticles and fluorescent molecules with DNA as tunable spacers.

Using stoichiometrically controlled 1:1 functionalization of gold nanoparticles with fluorescent dye molecules in which the dye molecule is held away from the particle surface by a rigid DNA spacer allows precise determination of the distance-dependent effect of the metal nanoparticles on fluorescence intensity. Two dyes were studied, Cy3 and Cy5, with two sizes of nanoparticles, 5 and 10 nm. The larger the particle, the more quenching of the photoluminescence (PL) intensity, due to increased overlap of the dye's emission spectrum with the Au surface plasmon resonance. Fluorescence is quenched significantly for distances somewhat larger than the particle diameter, in good agreement with the predictions of an electrodynamics model based on interacting dipoles. The distance dependence of surface energy transfer behavior, i.e. quenching efficiency, is proportional to 1/d(4), which involves no consideration of the size of the particle and the spectral overlap of the dye and AuNp. This surface energy transfer model is found qualitatively and agrees with the electrodynamic model, though the exponent is greater than 4 for the smaller nanoparticles (5 nm), and smaller than 4 for the larger nanoparticles (10 nm).

Distance dependent interhelical couplings of organic rods incorporated in DNA 4-helix bundles.

The synthesis of a conjugated linear organic module containing terminal salicylaldehyde groups and a central activated ester, designed for conjugation to amino-modified oligonucleotides, is presented. The organic module has a phenylene-ethynylene backbone and is highly fluorescent. It is conjugated to oligonucleotide sequences and incorporated into specific locations in a well-defined DNA 4-helix bundle (4-HB). The DNA-nanostructure offers precise location control of the organic modules which allows for selective interhelical coupling reactions. In this study, metal-salen formation as well as dihydrazone formation are used to covalently interlink the organic modules. Both coupling reactions are highly dependent on the distances between the organic modules in the 4-HB. Neighboring modules dimerize easier, whereas more distanced modules are less prone to react, even when the linkers are extended. The dimeric products are characterized by denaturing polyacrylamide gel electrophoresis (PAGE), high performance liquid chromatography (HPLC), and matrix assisted laser desorption/absorption ionization time-of-flight (MALDI TOF) mass spectrometry.

Control of self-assembly of DNA tubules through integration of gold nanoparticles.

The assembly of nanoparticles into three-dimensional (3D) architectures could allow for greater control of the interactions between these particles or with molecules. DNA tubes are known to form through either self-association of multi-helix DNA bundle structures or closing up of 2D DNA tile lattices. By the attachment of single-stranded DNA to gold nanoparticles, nanotubes of various 3D architectures can form, ranging in shape from stacked rings to single spirals, double spirals, and nested spirals. The nanoparticles are active elements that control the preference for specific tube conformations through size-dependent steric repulsion effects. For example, we can control the tube assembly to favor stacked-ring structures using 10-nanometer gold nanoparticles. Electron tomography revealed a left-handed chirality in the spiral tubes, double-wall tube features, and conformational transitions between tubes.

Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding.

An important goal of nanotechnology is to assemble multiple molecules while controlling the spacing between them. Of particular interest is the phenomenon of multivalency, which is characterized by simultaneous binding of multiple ligands on one biological entity to multiple receptors on another. Various approaches have been developed to engineer multivalency by linking multiple ligands together. However, the effects of well-controlled inter-ligand distances on multivalency are less well understood. Recent progress in self-assembling DNA nanostructures with spatial and sequence addressability has made deterministic positioning of different molecular species possible. Here we show that distance-dependent multivalent binding effects can be systematically investigated by incorporating multiple-affinity ligands into DNA nanostructures with precise nanometre spatial control. Using atomic force microscopy, we demonstrate direct visualization of high-affinity bivalent ligands being used as pincers to capture and display protein molecules on a nanoarray. These results illustrate the potential of using designer DNA nanoscaffolds to engineer more complex and interactive biomolecular networks.

Toward reliable gold nanoparticle patterning on self-assembled DNA nanoscaffold.

Assembly of gold nanoparticles (AuNP) into designer architectures with reliablity is important for nanophotonics and nanoelectronics applications. Toward this goal we present a new strategy to prepare AuNPs monofunctionalized with lipoic acid modified DNA oligos. This strategy offers increased bonding strength between DNA oligos and AuNP surface. These conjugates are further selectively mixed with other DNA strands and assembled into fixed sized DNA nanostructures carring a discrete number of AuNPs at desired positions. Atomic force microscopy imaging reveals a dramatically improved yield of the AuNPs on DNA tile structure compared to the ensembles using monothiolate AuNP-DNA conjugates.

A facile in situ generation of dithiocarbamate ligands for stable gold nanoparticle-oligonucleotide conjugates.

Here we demonstrate a facile strategy of preparing gold nanoparticle (AuNP) and DNA conjugates by in situ generation of strong metal affinity capping ligands, dithiocarbamates (DTC) modified oligonucleotides; the conjugates produced are stable at elevated temperature, resistant to ligand displacement and preserve the functionality of the conjugated oligonucleotides.

pH-driven conformational switch of "i-motif" DNA for the reversible assembly of gold nanoparticles.

Herein we demonstrate that gold nanoparticles conjugated to "i-motif" DNA behave like a pH dependent switch that undergoes reversible aggregations which can be easily visualized by the naked eye.

Patterning metallic nanoparticles by DNA scaffolds.

In order to miniaturize nanoelectronic circuitry and architectures, organizing the nanoelectronic components in deliberately designed complex patterns is desired. Bio-mimetic self-assembly is a possible approach to achieve this goal. Bio-macromolecules can serve as scaffolds to template the nanoelectronic components into patterns with precise periodicity and complexity. In this review, we will summarize the progress in organizing metallic nanoparticles templated by DNA scaffolds into one and two dimensional architectures.

Addressable molecular tweezers for DNA-templated coupling reactions.

Here we report the construction of fully addressable DNA-based molecular tweezers to actuate coupling reactions in a programmable fashion. Three tweezers, each bearing two coupling reactants, are self-assembled on a linear DNA track. A fourth tweezer floating freely in solution can be brought to any one of the tweezers and close them by the addition of a unique pair of "fuel" DNA strands. The coupling reactions happen when the tweezers are closed, and this can be controlled sequentially from one tweezer to another. A molecular device of this kind would not only enable programmable chemical reactions but also allow distance-dependent control of biomolecular interactions.

A modified strategy for Pictet-Spengler reaction leading to the synthesis of imidazoquinoxalines on solid phase.

An alternative strategy for Pictet-Spengler reaction involving an N-1 linked aromatic amine of imidazole and aldehydes is described. This is in contrast to a typical Pictet-Spengler reaction, which involves an aliphatic amine attached to the carbon of an activated aromatic nucleus and an aldehyde. Our strategy commences with the nucleophilic replacement of fluorine in resin-bound o-fluoro-nitrobenzoic acid with mono- or disubstituted imidazole, followed by reduction of the nitro group to give an N1 linked aromatic amine of the resin-bound imidazole. This was subsequently treated with an aldehyde in toluene at 80 degrees C and then oxidized in the presence of DDQ to give resin-bound imidazoquinoxalines. Finally, acidolytic cleavage furnished the desired compounds in high yields and purities.