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. However, activity of these oligonucleotides in brain tumors remains inadequately characterized. In this study, we developed a quantitative method to differentiate oligonucleotide-induced gene silencing in orthotopic GBM xenografts from gene silencing in normal brain tissue, and used this method to test the differential silencing activity of a chemically diverse panel of oligonucleotides. We show that oligonucleotides chemically optimized for pharmacological activity in normal brain tissue do not show consistent activity in GBM xenografts. We then survey multiple advanced oligonucleotide chemistries for their activity in GBM xenografts. Attaching lipid conjugates to oligonucleotides improves silencing in GBM cells across several different lipid classes. Highly hydrophobic lipid conjugates cholesterol and docosanoic acid enhance silencing but at the cost of higher neurotoxicity. Moderately hydrophobic, unsaturated fatty acid and amphiphilic lipid conjugates still improve activity without compromising safety. These oligonucleotide conjugates show promise for treating glioblastoma.

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. A library of protein mimics with different lengths, sequences, and heptad registers has been efficiently constructed. DNA-templated protein mimics show an α-helix or coiled-coil motif formation even when they are constructed from weakly interacting peptide units. Their attached DNA handles can be used to exert dynamic control over the protein mimics' secondary and tertiary structures. This modular strategy will facilitate the development of DNA-encoded protein libraries for the rapid discovery of new therapeutics, enzymes, and antibody mimics.

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. We report that this motif is exceptionally resistant to nucleases, with the constituent strands surviving for up to 1 month in the presence of serum. The conferred stability extends to adjacent non-polyA sequences, albeit with diminishing returns relative to their polyA sections due to hypothesized steric clashes. We introduce a strategy to regenerate stability through the introduction of free polyA strands or positively charged amino side chains, enhancing the stability of sequences of varied lengths. The proposed protection mechanism involves enzyme failure to recognize the unnatural polyA/CA motif, coupled with the motif's propensity to form long, bundling supramolecular fibers. The methodology provides a fundamentally new mechanism to protect nucleic acids from degradation using a supramolecular approach and increases lifetime in serum to days, weeks, or months.

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. We demonstrate that all approaches can achieve nuclease protection; however, the levels of DNA off-target crosslinking and damage vary. We then describe mild irradiation conditions intended to safeguard DNA against nuclease degradation. We demonstrate up to 25× increase in serum stability while minimizing off-target damage and maintaining functions such as hybridization efficiency, gene silencing, aptamer binding, and DNA nanostructure formation. Our methodology requires no complex instruments beyond a UV light source and no synthetic modification of the DNA itself, allowing for applications in numerous areas of nucleic acid therapy and nanotechnology.

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. However, because they maintain their structure by non-covalent interactions, they might suffer from disassembly in biologically relevant conditions, especially with regard to their interaction with serum proteins. Here, we report a systematic study of the factors that govern the fate of self-assembled SNAs. Varying the core chemistry and using stimuli-responsive disulfide crosslinking, we show that extracellular stability upon binding with serum proteins is important for recognition by membrane receptors, triggering cellular uptake. At the same time, intracellular dissociation is required for efficient therapeutic release. Disulfide-crosslinked SNAs combine these two properties and result in efficient and non-toxic unaided gene silencing therapeutics. We anticipate these investigations will help the translation of promising self-assembled structures towards in vivo gene silencing applications.

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. This interaction is crucial to sheet formation, marked by a chiral "signature", and sensitive to DNA sequence, where nanosheets form with a mixed sequence, but not with a homogeneous poly(thymine) sequence. This work opens the possibility of forming well-ordered, bilayer-like assemblies using a single DNA amphiphile for applications in cell sensing, nucleic acid therapeutic delivery and enzyme arrays.

Minimalist Design of Wireframe DNA Nanotubes: Tunable Geometry, Size, Chirality, and Dynamics.

DNA nanotubes (NTs) have attracted extensive interest as artificial cytoskeletons for biomedical, synthetic biology, and materials applications. Here, we report the modular design and assembly of a minimalist yet robust DNA wireframe nanotube with tunable cross-sectional geometry, cavity size, chirality, and length, while using only four DNA strands. We introduce an h-motif structure incorporating double-crossover (DX) tile-like DNA edges to achieve structural rigidity and provide efficient self-assembly of h-motif-based DNA nanotube (H-NT) units, thus producing programmable, micrometer-long nanotubes. We demonstrate control of the H-NT nanotube length via short DNA modulators. Finally, we use an enzyme, RNase H, to take these structures out of equilibrium and trigger nanotube assembly at a physiologically relevant temperature, underlining future cellular applications. The minimalist H-NTs can assemble at near-physiological salt conditions and will serve as an easily synthesized, DNA-economical modular template for biosensors, plasmonics, or other functional materials and as cost-efficient drug-delivery vehicles for biomedical applications.

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. The secondary structure of poly(adenine) DNA strands drives a temperature-dependent polymerization and assembly mechanism: copolymers stored in an SNA reservoir form fibers after thermal activation, which then aggregate upon cooling to form interwoven networks. DNA is often used as a programming code that aids in nanostructure addressability and function. Here, we show that the inherent physical and chemical properties of single-stranded DNA sequences also make them an ideal material to direct self-assembled morphologies and select for new methods of supramolecular polymerization.

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. Unlike conventional DNA nanostructure functionalization, which typically end-labels DNA strands, our approach incorporates functional groups into DNA with high density using small molecules and results in new DNA triple helices coated with alkylamine or alcohol units that grow into micrometer-long fibers. We find that small changes in the small molecule functional group can result in large structural and energetic variation in the overall assembly. A combination of circular dichroism, atomic force microscopy, molecular dynamics simulations, and a new thermodynamic method, transient equilibrium mapping, elucidated the molecular factors behind these large changes. In particular, we identify substantial DNA sugar and phosphate group deformations to accommodate a hydrogen bond between the phosphate and the small-molecule functional groups, as well as a critical chain length of the functional group which switches this interaction from intra- to interfiber. These parameters allow the controlled formation of hierarchical, hybrid DNA assemblies simply through the addition and variation of small, functionalized molecules.

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. The mechanical strength of the thymine-melamine-thymine triplet surpasses that of adenine-thymine base pairs, which enables a sensitive detection of melamine at 3 pM. The poly(thymine)-melamine duplex is orthogonal to native DNA base pairing and can undergo strand displacement without the need for overhangs. Its incorporation into two-dimensional grids and hybrid DNA-small-molecule polymers highlights the poly(thymine)-melamine duplex as an additional tool for DNA nanotechnology.

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. We can use single-molecule techniques - ranging from electron and fluorescence microscopies to electrical and force spectroscopies - to report on the structure, morphology, robustness, sample heterogeneity and other properties of these DNA nanoconstructs. In this Tutorial Review, we will detail how complementarity between static and dynamic single-molecule techniques can provide a unified image of DNA nanoarchitectures. The single-molecule methods that we discuss provide unprecedented insight into chemical and structural behaviour, yielding not just an average outcome but reporting on the distribution of values, ultimately showing how bulk properties arise from the collective behaviour of individual structures. As the fields of both DNA nanotechnology and single-molecule characterisation intertwine, a feedback loop is generated between disciplines, providing new opportunities for the development and operation of DNA-based materials as sensors, delivery vehicles, machinery and structural scaffolds.

Advancing Wireframe DNA Nanostructures Using Single-Molecule Fluorescence Microscopy Techniques.

DNA nanotechnology relies on the molecular recognition properties of DNA to produce complex architectures through self-assembly. The resulting DNA nanostructures allow scientists to organize functional materials at the nanoscale and have therefore found applications in many domains of materials science over the past several years. These scaffolds have been used to position proteins, nanoparticles, carbon nanotubes, and other nanomaterials with high spatial resolution. In addition to their remarkable performance as frameworks for other species, DNA constructs also possess interesting dynamic properties, which have led to their use in logic circuits, drug delivery vehicles, and molecular walkers. Although DNA nanostructures have become increasingly complex, the development of tools to study them has lagged. Currently, gel electrophoresis, dynamic light scattering, and ensemble fluorescence measurements are widely used to characterize DNA-based assemblies. Unfortunately, ensemble averaging in these methods obscures malformed structures and may mask properties associated with structure, length, and shape in polydisperse samples. While atomic force microscopy allows for the determination of morphology at the single-molecule level, this technique cannot typically be used to assess the dynamic properties of these constructs. To analyze the function of DNA-based devices such as molecular motors and reconfigurable nanostructures in real time, new single-molecule techniques are required. This Account details the work from our laboratories toward developing single-molecule fluorescence (SMF) methodologies for the structural and dynamic characterization of wireframe DNA nanostructures, one at a time. The methods described herein provide us with two separate yet related sets of information: First, we can statically examine the nanostructures one by one to assess their robustness, structural fidelity, and morphology. This is primarily done using two-color stepwise photobleaching, wherein we can examine the subunit stoichiometry of our assemblies before and after various perturbations to the structures. For example, we can introduce length mismatches to cause the nanotube to bend or perform strand displacement reactions to generate single-stranded, flexible analogues of our materials. Second, due to the unmatched spatiotemporal resolution of SMF techniques, we can study the dynamic character of these assemblies by implementing structural changes to the nanotube and monitoring them in real time. With this structural and dynamic information in hand, our groups have additionally developed new tools for the improved construction of DNA nanotubes, inspired by solid-phase DNA synthesis. By assembling the nanotubes in a stepwise manner, highly monodisperse nanostructures of any desired length can be made without a template strand. In this way, unique building blocks can also be added sequence-specifically, allowing for the production of user-defined scaffolds to organize nanoscale materials in three dimensions. This method, in combination with our imaging and analysis protocols, may be extended to assemble and inspect other supramolecular constructs in a controlled manner. Overall, by combining synthesis, characterization, and analysis, these single-molecule techniques hold the potential to advance the study of DNA nanostructures and dynamic DNA-based devices.

Target Self-Enhanced Selectivity in Metal-Specific DNAzymes.

Highly selective recognition of metal ions by rational ligand design is challenging, and simple metal binding by biological ligands is often obscured by nonspecific interactions. In this work, binding-triggered catalysis is used and metal selectivity is greatly increased by increasing the number of metal ions involved, as exemplified in a series of in vitro selected RNA-cleaving DNAzymes. The cleavage junction is modified with a glycyl-histidine-functionalized tertiary amine moiety to provide multiple potential metal coordination sites. DNAzymes that bind 1, 2, and 3 Zn2+ ions, increased their selectivity for Zn2+ over Co2+ ions from approximately 20-, 1000-, to 5000-fold, respectively. This study offers important insights into metal recognition by combining rational ligand design and combinatorial selection, and it provides a set of new DNAzymes with excellent selectivity for Zn2+ ions.

Amplified Self-Immolative Release of Small Molecules by Spatial Isolation of Reactive Groups on DNA-Minimal Architectures.

Triggering the release of small molecules in response to unique biomarkers is important for applications in drug delivery and biodetection. Due to low quantities of biomarker, amplifying release is necessary to gain appreciable responses. Nucleic acids have been used for both their biomarker-recognition properties and as stimuli, notably in amplified small-molecule release by nucleic-acid-templated catalysis (NATC). The multiple components and reversibility of NATC, however, make it difficult to apply in vivo. Herein, we report the use of the hybridization chain reaction (HCR) for the amplified, conditional release of small molecules from standalone nanodevices. We couple HCR with a DNA-templated reaction resulting in the amplified, immolative release of small molecules. We integrate the HCR components into single nanodevices as DNA tracks and spherical nucleic acids, spatially isolating reactive groups until triggering. This could be applied to biosensing, imaging, and drug delivery.

Transition-Metal-Functionalized DNA Double-Crossover Tiles: Enhanced Stability and Chirality Transfer to Metal Centers.

The double crossover junction (DX) is a fundamental building block for generating complex and varied structures from DNA. However, its implementation in functional devices is limited to the inherent properties of DNA itself. Here, we developed design strategies to generate the first metal-DX DNA tiles (DXM ) by site-specifically functionalizing the tile crossovers with tetrahedral binding pockets that coordinate CuI . These DX junctions bind two CuI ions independently at distinct sites, display greater thermal stability than native DX tiles upon metalation, and melt in a cooperative fashion. In addition, the right-handed helical chirality of DNA is transferred to the metal centers. Our tiles display high metal ion selectivity, such that CuII is spontaneously reduced to CuI in situ. By modifying our design over three generations of tiles, we elucidated the thermodynamic and geometric requirements for the successful assembly of DXM tiles, which have direct applicability in developing robust, stable DNA-based materials with electroactive, photoactive, and catalytic properties.

Spatial Presentation of Cholesterol Units on a DNA Cube as a Determinant of Membrane Protein-Mimicking Functions.

Cells use membrane proteins as gatekeepers to transport ions and molecules, catalyze reactions, relay signals, and interact with other cells. DNA nanostructures with lipidic anchors are promising as membrane protein mimics because of their high tunability. However, the design features specifying DNA nanostructures' functions in lipid membranes are yet to be fully understood. Here, we show that altering patterns of cholesterol units on a cubic DNA scaffold dramatically changes its interaction mode with lipid membranes. This results in simple design rules that allow a single DNA nanostructure to reproduce multiple membrane protein functions: peripheral anchoring, nanopore behavior, and conformational switching to reveal membrane-binding units. Strikingly, the DNA-cholesterol cubes constitute the first open-walled DNA nanopores, as only a quarter of their wall is made of DNA. This functional diversity can increase our fundamental understanding of membrane phenomena and result in sensing, drug delivery, and cell manipulation tools.

"Printing" DNA Strand Patterns on Small Molecules with Control of Valency, Directionality, and Sequence.

The incorporation of synthetic molecules as corner units in DNA structures has been of interest over the last two decades. In this work, we present a facile method for generating branched small molecule-DNA hybrids with controllable valency, different sequences, and directionalities (5'-3') using a "printing" process from a simple 3-way junction structure. We also show that the DNA-imprinted small molecule can be extended asymmetrically using polymerase chain reaction (PCR) and can be replicated chemically. This strategy provides opportunities to achieve new structural motifs in DNA nanotechnology and introduce new functionalities to DNA nanostructures.

Cyanine-Mediated DNA Nanofiber Growth with Controlled Dimensionality.

Supramolecular one-dimensional (1D) architectures are of high interest in drug delivery and templation of complex linear arrays due to their high aspect ratio and rigidity. A particular desire is the access of 1D nanostructures with high functionality and biorelevance, which opens the door to  their applications in materials science and nanomedicine. Here we report the discovery that the site-specific introduction of a cyanine (Cy3) dye unit in sequence-defined DNA amphiphiles causes a complete shift of the overall structure from spheres to 1D DNA nanofibers in aqueous media. We show that the generation of DNA nanofibers is dependent on the presence of cyanine units and their position within the DNA-polymer hybrid. We further demonstrate an example of stimuli-responsive shape-shifting DNA nanofibers to highlight the role of the dye in the overall assembly. Notably, we show the preparation of fibers with controlled length by seeded-growth mechanism. Additionally, the DNA nanofibers exhibit a change in Cy3 dye optical properties upon assembly, typical of cyanine dye aggregation, which can be used to monitor the fiber growth process. To demonstrate the functionality of these structures, we show the templation of gold nanoparticles (AuNP) along the fiber length and demonstrate the directional templation of DNA nanofibers on rectangular DNA origami. Our findings provide a method for generating functional nanomaterials and hierarchical complex architectures and show promise as a platform for biosensing and targeted drug delivery.

Encapsulation of Gold Nanoparticles into DNA Minimal Cages for 3D-Anisotropic Functionalization and Assembly.

Gold nanoparticles (AuNPs) endowed with anisotropic DNA valency are an important class of materials, as they can assemble into complex structures with a minimal number of DNA strands. However, methods to encode 3D DNA strand patterns on AuNPs with a controlled number of unique DNA strands in a predesigned spatial arrangement remain elusive. In this work, a simple one-step method to yield such DNA-decorated AuNPs is demonstrated, through encapsulating AuNPs into DNA minimal nanocages. The AuNP@DNA cage encapsulation complex inherits the 3D anisotropic molecular information from the DNA nanocage with enhanced structural stability. The DNA nanocage can be further functionalized and used as a building block for the self-assembly of complex architectures, such as dimers and trimers, programmed assemblies with sequential growth DNA backbones and DNA origami.

Modular Strategy To Expand the Chemical Diversity of DNA and Sequence-Controlled Polymers.

Sequence-defined polymers with customizable sequences, monodispersity, substantial length, and large chemical diversity are of great interest to mimic the efficiency and selectivity of biopolymers. We report an efficient, facile, and scalable synthetic route to introduce many chemical functionalities, such as amino acids and sugars in nucleic acids and sequence-controlled oligophosphodiesters. Through achiral tertiary amine molecules that are perfectly compatible with automated DNA synthesis, readily available amines or azides can be turned into phosphoramidites in two steps only. Individual attachment yields on nucleic acids and artificial oligophosphodiesters using automated solid-phase synthesis (SPS) were >90% in almost all cases. Using this method, multiple water-soluble sequence-defined oligomers bearing a range of functional groups in precise sequences could be synthesized and purified in high yields. The method described herein significantly expands the library of available functionalities for nucleic acids and sequence-controlled polymers.