Thomas G. W. Edwardson

Transfer of molecular recognition information from DNA nanostructures to gold nanoparticles

DNA nanotechnology offers unparalleled precision and programmability for the bottom-up organization of materials. This approach relies on pre-assembling a DNA scaffold, typically containing hundreds of different strands, and using it to position functional components. A particularly attractive strategy is to employ DNA nanostructures not as permanent scaffolds, but as transient, reusable templates to transfer essential information to other materials. To our knowledge, this approach, akin to top-down lithography, has not been examined. Here we report a molecular printing strategy that chemically transfers a discrete pattern of DNA strands from a three-dimensional DNA structure to a gold nanoparticle. We show that the particles inherit the DNA sequence configuration encoded in the parent template with high fidelity. This provides control over the number of DNA strands and their relative placement, directionality and sequence asymmetry. Importantly, the nanoparticles produced exhibit the site-specific addressability of DNA nanostructures, and are promising components for energy, information and biomedical applications. DNA nanostructures are typically used as molecular scaffolds. Now, it has been shown that they can also act as reusable templates for ‘molecular printing’ of DNA strands onto gold nanoparticles. The products inherit the recognition elements of the parent template: number, orientation and sequence asymmetry of DNA strands. This converts isotropic nanoparticles into complex building blocks.

An Efficient and Modular Route to Sequence‐Defined Polymers Appended to DNA

Inspired by biological polymers, sequence-controlled synthetic polymers are highly promising materials that integrate the robustness of synthetic systems with the information-derived activity of biological counterparts. Polymer-biopolymer conjugates are often targeted to achieve this union; however, their synthesis remains challenging. We report a stepwise solid-phase approach for the generation of completely monodisperse and sequence-defined DNA-polymer conjugates using readily available reagents. These polymeric modifications to DNA display self-assembly and encapsulation behavior-as evidenced by HPLC, dynamic light scattering, and fluorescence studies-which is highly dependent on sequence order. The method is general and has the potential to make DNA-polymer conjugates and sequence-defined polymers widely available.

Precision Polymers and 3D DNA Nanostructures: Emergent Assemblies from New Parameter Space

Polymer self-assembly and DNA nanotechnology have both proved to be powerful nanoscale techniques. To date, most attempts to merge the fields have been limited to placing linear DNA segments within a polydisperse block copolymer. Here we show that, by using hydrophobic polymers of a precisely predetermined length conjugated to DNA strands, and addressable 3D DNA prisms, we are able to effect the formation of unprecedented monodisperse quantized superstructures. The structure and properties of larger micelles-of-prisms were probed in depth, revealing their ability to participate in controlled release of their constituent nanostructures, and template light-harvesting energy transfer cascades, mediated through both the addressability of DNA and the controlled aggregation of the polymers.

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.

“DNA–Teflon” sequence-controlled polymers

Perfluorocarbons (PFCs) are a promising class of molecules for medical applications: they are detectable through 19F nuclear magnetic resonance (NMR) and they assemble separately from water or lipophilic phases, thus bringing unique supramolecular interactions into nanostructures. We report the ready insertion of PFCs into nucleic acids, as well as non-natural polymers in a sequence-defined fashion. This is achieved via an automated and efficient synthetic pathway using phosphoramidite chemistry. Modulating the PFC tail length of “DNA–Teflon” block copolymers resulted in micelles that are almost monodisperse, have a low critical micelle concentration (CMC), are traceable by 19F NMR and are responsive to external stimuli. Strong fluorine–fluorine interactions in DNA duplexes allowed remarkable melting temperature increases and provided nuclease resistance. Finally, PFC insertion into siRNA was achieved, and the conjugates were efficient for gene silencing, outlining that these modifications are highly suitable for oligonucleotide therapeutics and bioimaging tools.

Development of DNA Nanostructures for High-Affinity Binding to Human Serum Albumin

The development of nucleic acid therapeutics has been hampered by issues associated with their stability and in vivo delivery. To address these challenges, we describe a new strategy to engineer DNA structures with strong binding affinity to human serum albumin (HSA). HSA is the most abundant protein in the blood and has a long circulation half-life (19 days). It has been shown to hinder phagocytosis, is retained in tumors, and aids in cellular penetration. Indeed, HSA has already been successfully used for the delivery of small-molecule drugs and nanoparticles. We show that conjugating dendritic alkyl chains to DNA creates amphiphiles that exhibit high-affinity (Kd in low nanomolar range) binding to HSA. Notably, complexation with HSA did not hinder the activity of silencing oligonucleotides inside cells, and the degradation of DNA strands in serum was significantly slowed. We also show that, in a site-specific manner, altering the number and orientation of the amphiphilic ligand on a self-assembled DNA nanocube can modulate the affinity of the DNA cage to HSA. Moreover, the serum half-life of the amphiphile bound to the cage and the protein was shown to reach up to 22 hours, whereas unconjugated single-stranded DNA was degraded within minutes. Therefore, adding protein-specific binding domains to DNA nanostructures can be used to rationally control the interface between synthetic nanostructures and biological systems. A major challenge with nanoparticles delivery is the quick formation of a protein corona (i.e., protein adsorbed on the nanoparticle surface) upon injection to biological media. We foresee such DNA cage-protein complexes as new tools to study the role of this protein adsorption layer with important implications in the efficient delivery of RNAi therapeutics in vitro and in vivo.

Modular Protein Cages for Size-Selective RNA Packaging in Vivo

Protein cages have recently emerged as an important platform for nanotechnology development. Of the naturally existing protein cages, viruses are among the most efficient nanomachines, overcoming various barriers to achieve component replication and efficient self-assembly in complex biological milieu. We have designed an artificial system that can carry out the most basic steps of viral particle assembly in vivo. Our strategy is based on patchwork capsids formed from Aquifex aeolicus lumazine synthase and a circularly permuted variant with appended cationic peptides. These two-component protein containers self-assemble in vivo, capturing endogenous RNA molecules in a size-selective manner. By varying the number and design of the RNA-binding peptides displayed on the lumenal surface, the length of guest RNA can be further controlled. Using a fluorescent aptamer, we also show that short-lived RNA species are captured by the protein cage. This platform has potential as a model system for investigating virus assembly, as well as developing RNA regulation or sampling tools to augment biotechnology.

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.