Toward Automated DNA Nanoprinting: Advancing the Synthesis of Covalently Branched DNA.

Covalently branched DNA molecules are hybrid structures where a small molecule core is covalently linked to different DNA strands. They merge the programmability of DNA nanotechnology with synthetic molecules' functionality, offering enhanced stability over their non-covalent counterparts like double-crossover tiles. They enable the efficient assembly of stable DNA nanostructures with new geometries and functionalities.

Automated Synthesis of DNA Nanostructures.

DNA nanotechnology has revolutionized the ability to position matter at the nanoscale, but the preparation of DNA-based architectures remains laborious. To facilitate the formation of custom structures, a fully automated method is reported to produce sequence- and size-defined DNA nanotubes. By programming the sequential addition of desired building blocks, rigid DX-tile-based DNA nanotubes and flexible wireframe DNA structures are attained, where the total number of possible constructs increases as a power function of the number of different units available.

DNA Hierarchical Superstructures from Micellar Units: Stiff Hydrogels and Anisotropic Nanofibers.

Supramolecular materials have been assembled using a wide range of interactions, including the hydrophobic effect, DNA base-pairing, and hydrogen bonding. Specifically, DNA amphiphiles with a hydrophobic building block self-assemble into diverse morphologies depending on the length and composition of both blocks. Herein, we take advantage of the orthogonality of different supramolecular interactions - the hydrophobic effect, Watson-Crick-Franklin base pairing and RNA kissing loops - to create hierarchical self-assemblies with controlled morphologies on both the nanometer and the micrometer scales.

Heat-activated growth of metastable and length-defined DNA fibers expands traditional polymer assembly.

Biopolymers such as nucleic acids and proteins exhibit dynamic backbone folding, wherein site-specific intramolecular interactions determine overall structure. Proteins then hierarchically assemble into supramolecular polymers such as microtubules, that are robust yet dynamic, constantly growing or shortening to adjust to cellular needs. The combination of dynamic, energy-driven folding and growth with structural stiffness and length control is difficult to achieve in synthetic polymer self-assembly.

Quantifying the activity profile of ASO and siRNA conjugates in glioblastoma xenograft tumors in vivo.

Glioblastoma multiforme is a universally lethal brain tumor that largely resists current surgical and drug interventions. Despite important advancements in understanding GBM biology, the invasiveness and heterogeneity of these tumors has made it challenging to develop effective therapies. Therapeutic oligonucleotides-antisense oligonucleotides and small-interfering RNAs-are chemically modified nucleic acids that can silence gene expression in the brain.

Exceptional Nuclease Resistance of DNA and RNA with the Addition of Small-Molecule Nucleobase Mimics.

Nucleases present a formidable barrier to the application of nucleic acids in biology, significantly reducing the lifetime of nucleic acid-based drugs. Here, we develop a novel methodology to protect DNA and RNA from nucleases by reconfiguring their supramolecular structure through the addition of a nucleobase mimic, cyanuric acid. In the presence of cyanuric acid, polyadenine strands assemble into triple helical fibers known as the polyA/CA motif.

Control of the Assembly and Disassembly of Spherical Nucleic Acids Is Critical for Enhanced Gene Silencing.

Spherical nucleic acids─nanospheres with nucleic acids on their corona─have emerged as a promising class of nanocarriers, aiming to address the shortcomings of traditional nucleic therapeutics, namely, their poor stability, biodistribution, and cellular entry. By conjugating hydrophobic monomers to a growing nucleic acid strand in a sequence-defined manner, our group has developed self-assembled spherical nucleic acids (SaSNAs), for unaided, enhanced gene silencing. By virtue of their self-assembled nature, SaSNAs can disassemble under certain conditions in contrast to covalent or gold nanoparticle SNAs.

DNA-Mediated Peptide Assembly into Protein Mimics.

The design of new protein structures is challenging due to their vast sequence space and the complexity of protein folding. Here, we report a new modular DNA-templated strategy to construct protein mimics. We achieve the spatial control of multiple peptide units by conjugation with DNA and hybridization to a branched DNA trimer template followed by covalent stapling of the preorganized peptides into a single unit.

Dendritic amphiphilic siRNA: Selective albumin binding, efficacy, and low toxicity.

Although an increasing number of small interfering RNA (siRNA) therapies are reaching the market, the challenge of efficient extra-hepatic delivery continues to limit their full therapeutic potential. Drug delivery vehicles and hydrophobic conjugates are being used to overcome the delivery bottleneck. Previously, we reported a novel dendritic conjugate that can be appended efficiently to oligonucleotides, allowing them to bind albumin with nanomolar affinity.

Impact of the Core Chemistry of Self-Assembled Spherical Nucleic Acids on their In Vitro Fate.

Nucleic acid therapeutics (NATs), such as mRNA, small interfering RNA or antisense oligonucleotides are extremely efficient tools to modulate gene expression and tackle otherwise undruggable diseases. Spherical nucleic acids (SNAs) can efficiently deliver small NATs to cells while protecting their payload from nucleases, and have improved biodistribution and muted immune activation. Self-assembled SNAs have emerged as nanostructures made from a single DNA-polymer conjugate with similar favorable properties as well as small molecule encapsulation.

Sequence-Controlled Spherical Nucleic Acids: Gene Silencing, Encapsulation, and Cellular Uptake.

Antisense oligonucleotides (ASOs) can predictably alter RNA processing and control protein expression; however, challenges in the delivery of these therapeutics to specific tissues, poor cellular uptake, and endosomal escape have impeded progress in translating these agents into the clinic. Spherical nucleic acids (SNAs) are nanoparticles with a DNA external shell and a hydrophobic core that arise from the self-assembly of ASO strands conjugated to hydrophobic polymers. SNAs have recently shown significant promise as vehicles for improving the efficacy of ASO cellular uptake and gene silencing.

Two-Dimensional Supramolecular Polymerization of DNA Amphiphiles is Driven by Sequence-Dependent DNA-Chromophore Interactions.

Two-dimensional (2D) assemblies of water-soluble block copolymers have been limited by a dearth of systematic studies that relate polymer structure to pathway mechanism and supramolecular morphology. Here, we employ sequence-defined triblock DNA amphiphiles for the supramolecular polymerization of free-standing DNA nanosheets in water. Our systematic modulation of amphiphile sequence shows the alkyl chain core forming a cell membrane-like structure and the distal π-stacking chromophore block folding back to interact with the hydrophilic DNA block on the nanosheet surface.

Programming rigidity into size-defined wireframe DNA nanotubes.

Nanotubes built from DNA hold promise for several biological and materials applications, due to their high aspect ratio and encapsulation potential. A particularly appealing goal is to control the size, shape, and dynamic behaviour of DNA nanotubes with minimal design alteration, as nanostructures of varying morphologies and lengths have been shown to exhibit distinct cellular uptake, encapsulation behaviour, and biodistribution. Herein, we report a systematic investigation, combining experimental and computational design, to modulate the length, flexibility, and longitudinal patterns of wireframe DNA nanotubes.

Small Molecule-Templated DNA Hydrogel with Record Stiffness Integrates and Releases DNA Nanostructures and Gene Silencing Nucleic Acids.

Deoxyribonucleic acid (DNA) hydrogels are a unique class of programmable, biocompatible materials able to respond to complex stimuli, making them valuable in drug delivery, analyte detection, cell growth, and shape-memory materials. However, unmodified DNA hydrogels in the literature are very soft, rarely reaching a storage modulus of 10  Pa, and they lack functionality, limiting their applications. Here, a DNA/small-molecule motif to create stiff hydrogels from unmodified DNA, reaching 10  Pa in storage modulus is used.

Stabilization of Functional DNA Structures with Mild Photochemical Methods.

A significant barrier to biological applications of DNA structures is their instability to nucleases. UV-mediated thymine dimerization can crosslink and stabilize DNA nanostructures, but its effect on DNA strand hybridization fidelity and function is unclear. In this work, we first compare a number of methods for DNA irradiation with different wavelengths of light and different photosensitizers.

DNA Sequence and Length Dictate the Assembly of Nucleic Acid Block Copolymers.

The self-assembly of block copolymers is often rationalized by structure and microphase separation; pathways that diverge from this parameter space may provide new mechanisms of polymer assembly. Here, we show that the sequence and length of single-stranded DNA directly influence the self-assembly of sequence-defined DNA block copolymers. While increasing the length of DNA led to predictable changes in self-assembly, changing only the sequence of DNA produced three distinct structures: spherical micelles (spherical nucleic acids, SNAs) from flexible poly(thymine) DNA, fibers from semirigid mixed-sequence DNA, and networked superstructures from rigid poly(adenine) DNA.

Asymmetric patterning drives the folding of a tripodal DNA nanotweezer.

DNA tweezers have emerged as powerful devices for a wide range of biochemical and sensing applications; however, most DNA tweezers consist of single units activated by DNA recognition, limiting their range of motion and ability to respond to complex stimuli. Herein, we present an extended, tripodal DNA nanotweezer with a small molecule junction. Simultaneous, asymmetric elongation of our molecular core is achieved using polymerase chain reaction (PCR) to produce length- and sequence-specific DNA arms with repeating DNA regions.

Tuning DNA Supramolecular Polymers by the Addition of Small, Functionalized Nucleobase Mimics.

Nucleobase mimicking small molecules able to reconfigure DNA are a recently discovered strategy that promises to extend the structural and functional diversity of nucleic acids. However, only simple, unfunctionalized molecules such as cyanuric acid and melamine have so far been used in this approach. In this work, we show that the addition of substituted cyanuric acid molecules can successfully program polyadenine strands to assemble into supramolecular fibers.

A dissipative pathway for the structural evolution of DNA fibres.

Biochemical networks interconnect, grow and evolve to express new properties as different chemical pathways are selected during a continuous cycle of energy consumption and transformation. In contrast, synthetic systems that push away from equilibrium usually return to the same self-assembled state, often generating waste that limits system recyclability and prevents the formation of adaptable networks. Here we show that annealing by slow proton dissipation selects for otherwise inaccessible morphologies of fibres built from DNA and cyanuric acid.

Design and enhanced gene silencing activity of spherical 2'-fluoroarabinose nucleic acids (FANA-SNAs).

Drug delivery vectors for nucleic acid therapeutics (NATs) face significant barriers for translation into the clinic. Spherical nucleic acids (SNAs) - nanoparticles with an exterior shell made up of DNA strands and a hydrophobic interior - have recently shown great potential as vehicles to improve the biodistribution and efficacy of NATs. To date, SNA design has not taken advantage of the powerful chemical modifications available to NATs.

DNA Nanostructures: Current Challenges and Opportunities for Cellular Delivery.

DNA nanotechnology has produced a wide range of self-assembled structures, offering unmatched possibilities in terms of structural design. Because of their programmable assembly and precise control of size, shape, and function, DNA particles can be used for numerous biological applications, including imaging, sensing, and drug delivery. While the biocompatibility, programmability, and ease of synthesis of nucleic acids have rapidly made them attractive building blocks, many challenges remain to be addressed before using them in biological conditions.

A poly(thymine)-melamine duplex for the assembly of DNA nanomaterials.

The diversity of DNA duplex structures is limited by a binary pair of hydrogen-bonded motifs. Here we show that poly(thymine) self-associates into antiparallel, right-handed duplexes in the presence of melamine, a small molecule that presents a triplicate set of the hydrogen-bonding face of adenine. X-ray crystallography shows that in the complex two poly(thymine) strands wrap around a helical column of melamine, which hydrogen bonds to thymine residues on two of its three faces.

Single-molecule methods in structural DNA nanotechnology.

Single molecules can now be visualised with unprecedented precision. As the resolution of single-molecule experiments improves, so too does the breadth, quantity and quality of information that can be extracted using these methodologies. In the field of DNA nanotechnology, we use programmable interactions between nucleic acids to generate complex, multidimensional structures.