Reversible Ligation of Programmed DNA-Gold Nanoparticle Assemblies.

We demonstrate a new method to reversibly cross-link DNA-nanoparticle dimers, trimers, and tetramers using light as an external stimulus. A DNA interstrand photo-cross-linking reaction is possible via ligation of a cyano-vinyl carbazole nucleoside with an opposite thymine when irradiated at 365 nm. This reaction results in nanoparticle assemblies that are not susceptible to DNA dehybridization conditions. The chemical bond between the two complementary DNA strands can be reversibly broken upon light irradiation at 312 nm. This is the first example of reversible ligation in DNA-nanoparticle assemblies using light and enables new developments in the field of programmed nanoparticle organization.

Triplex-mediated analysis of cytosine methylation at CpA sites in DNA.

Modified triplex-forming oligonucleotides distinguish 5-methyl cytosine from unmethylated cytosine in DNA duplexes by differences in triplex melting temperatures. The discrimination is sequence-specific; dramatic differences in stabilisation are seen for CpA methylation, whereas CpG methylation is not detected. This direct detection of DNA methylation constitutes a new approach for epigenetic analysis.

A new modular approach to nanoassembly: stable and addressable DNA nanoconstructs via orthogonal click chemistries.

Thermodynamic instability is a problem when assembling and purifying complex DNA nanostructures formed by hybridization alone. To address this issue, we have used photochemical fixation and orthogonal copper-free, ring-strain-promoted, click chemistry for the synthesis of dimeric, trimeric, and oligomeric modular DNA scaffolds from cyclic, double-stranded, 80-mer DNA nanoconstructs. This particular combination of orthogonal click reactions was more effective for nanoassembly than others explored. The complex nanostructures are stable to heat and denaturation agents and can therefore be purified and characterized. They are addressable in a sequence-specific manner by triplex formation, and they can be reversibly and selectively deconstructed. Nanostructures utilizing this orthogonal, chemical fixation methodology can be used as building blocks for nanomachines and functional DNA nanoarchitectures.

Membrane protrusion coarsening and nanotubulation within giant unilamellar vesicles.

Hydrophobic side groups on a stimuli-responsive polymer, encapsulated within a single giant unilamellar vesicle, enable membrane attachment during compartment formation at elevated temperatures. We thermally modulated the vesicle through implementation of an IR laser via an optical fiber, enabling localized directed heating. Polymer-membrane interactions were monitored using confocal imaging techniques as subsequent membrane protrusions occurred and lipid nanotubes formed in response to the polymer hydrogel contraction. These nanotubes, bridging the vesicle membrane to the contracting hydrogel, were retained on the surface of the polymer compartment, where they were transformed into smaller vesicles in a process reminiscent of cellular endocytosis. This development of a synthetic vesicle system containing a stimuli-responsive polymer could lead to a new platform for studying inter/intramembrane transport through lipid nanotubes.

Self-assembled DNA-based fluorescence waveguide with selectable output.

Using the principle of self-assembly, a fluorescence-based photonic network is constructed with one input and two spatially and spectrally distinct outputs. A hexagonal DNA nanoassembly is used as a scaffold to host both the input and output dyes. The use of DNA to host functional groups enables spatial resolution on the level of single base pairs, well below the wavelength of light. Communication between the input and output dyes is achieved through excitation energy transfer. Output selection is achieved by the addition of a mediator dye intercalating between the DNA base pairs transferring the excitation energy from input to output through energy hopping. This creates a tool for selective excitation energy transfer on the nanometer scale with spectral and spatial control. The ability to direct excitation energy in a controlled way on the nanometer scale is important for the incorporation of photochemical processes in nanotechnology.

Peptidomimetic bond formation by DNA-templated acyl transfer.

The efficiencies of DNA-templated acyl transfer reactions between a thioester modified oligonucleotide and a series of amine and thiol based nucleophiles are directly compared. The reactivity of the nucleophile, reaction conditions (solvent, buffer, pH) and linker length all play important roles in determining the efficiency of the transfer reaction. Careful optimisation of the system enables the use of DNA-templated synthesis to form stable peptide-like bonds under mild aqueous conditions close to neutral pH.

CG base pair recognition within DNA triple helices by modified N-methylpyrrolo-dC nucleosides.

3-Aminophenyl-modified analogues of the bicyclic nucleoside N-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one were synthesised and incorporated directly into triplex-forming oligonucleotides in order to utilise their extended hydrogen bonding motif for recognition of the CG base pair. All analogues demonstrated strong binding affinity and very good selectivity for CG from pH 6.2 to 7.0; a marked improvement on previous modifications.

Programmed assembly of peptide-functionalized gold nanoparticles on DNA templates.

We present a novel nanoparticle building block system based on the interactions between short synthetic oligonucleotides and peptides. Gold nanoparticles coated with DNA-binding peptides can be attached to self-organized oligonucleotide templates to formulate well-ordered structures of nanoparticles. By regulating the amount of DNA-binding peptide attached to the nanoparticle surface and using specifically designed oligonucleotides, the nanoparticle assembly can be controlled to form dimers, trimers, and adjustable-length nanoparticle chains as well as more complex structures.

Potent triple helix stabilization by 5',3'-modified triplex-forming oligonucleotides.

Anthraquinone and pyrene analogues attached to the 3' and/or 5' termini of triplex-forming oligonucleotides (TFOs) by various linkers increased the stability of parallel triple helices. The modifications are simple to synthesize and can be introduced during standard solid-phase oligonucleotide synthesis. Potent triplex stability was achieved by using doubly modified TFOs, which in the most favourable cases gave an increase in melting temperature of 30 degrees C over the unmodified counterparts and maintained their selectivity for the correct target duplex. Such TFOs can produce triplexes with melting temperatures of 40 degrees C at pH 7 even though they do not contain any triplex-stabilizing base analogues. These studies have implications for the design of triplex-forming oligonucleotides for use in biology and nanotechnology.

CG base pair recognition within DNA triple helices using N-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues.

Triplex-mediated recognition of Py.Pu base pairs in DNA is a greater challenge than for Pu.Py base pairs as fewer hydrogen bonds are presented for binding in the major groove. Initial studies on m-aminophenyl-modified analogues of the bicyclic nucleoside N-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7 H)-one suggest that selective recognition of the CG base pair is possible.