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ConspectusStructural DNA nanotechnology offers a unique self-assembly toolbox to construct soft materials of arbitrary complexity, through bottom-up approaches including DNA origami, brick, wireframe, and tile-based assemblies. This toolbox can be expanded by incorporating interactions orthogonal to DNA base-pairing such as metal coordination, small molecule hydrogen bonding, π-stacking, fluorophilic interactions, or the hydrophobic effect. These interactions allow for hierarchical and long-range organization in DNA supramolecular assemblies through a DNA-minimal approach: the use of fewer unique DNA sequences to make complex structures.Here we describe our research group's work to integrate these orthogonal interactions into DNA and its supramolecular assemblies. Using automated solid phase techniques, we synthesized s equence-defined D NA p olymers (SDPs) featuring a wide range of functional groups, achieving high yields in the process. These SDPs can assemble into not only isotropic spherical morphologies─such as spherical nucleic acids (SNAs)─but also into anisotropic nanostructures such as 1D nanofibers and 2D nanosheets. Our structural and molecular modeling studies revealed new insights into intermolecular chain packing and intramolecular chain folding, influenced by phosphodiester positioning and SDP sequence. Using these new self-assembly paradigms, we created hierarchical, anisotropic assemblies and developed systems exhibiting polymorphism and chiroptical behavior dependent on the SDP sequence. We could also precisely control the size of our nanofiber assemblies via nucleation-growth supramolecular polymerization and create compartmentalized nanostructures capable of precise surface functionalization.The exquisite control over sequence, composition, and length allowed us to combine our SDPs with nanostructures including DNA wireframe assemblies such as prisms, nanotubes, and cubes to create hybrid, stimuli-responsive assemblies exhibiting emergent structural and functional modes. The spatial control of our assemblies enabled their use as nanoreactors for chemical transformations in several ways: via hybridization chain reaction within SNA coronas, through chemical conjugation within SNA cores, and through a molecular "printing" approach within wireframe assemblies for nanoscale information transfer and the creation of anisotropic "DNA-printed" polymer particles.We have also employed our SDP nanostructures toward biological and therapeutic applications. We demonstrated that our SNAs could serve as both extrinsic and intrinsic therapeutic platforms, with improved cellular internalization and biodistribution profiles, and excellent gene silencing activities. Using SDPs incorporating hydrophobic dendrons, high-affinity and highly specific oligonucleotide binding to human serum albumin was demonstrated. These structures showed an increased stability to nuclease degradation, reduced nonspecific cellular uptake, no toxicity even at high concentrations, and excellent biodistribution beyond the liver, resulting in unprecedented gene silencing activity in various tissues.Control over the sequence has thus presented us with a unique polymeric building block in the form of the SDP, which combines the chemical and structural diversity of polymers with the programmability of DNA. By linking these orthogonal assembly languages, we have discovered new self-assembly rules, created DNA-minimal nanostructures, and demonstrated their utility through a range of applications. Developing this work further will open new avenues in the fields of DNA nanomaterials, nucleic acid therapeutics, as well as block copolymer self-assembly.

Oligonucleotide therapeutics, including antisense oligonucleotides and small interfering RNA, offer promising avenues for modulating the expression of disease-associated proteins. However, challenges such as nuclease degradation, poor cellular uptake, and unspecific targeting hinder their application. To overcome these obstacles, spherical nucleic acids have emerged as versatile tools for nucleic acid delivery in biomedical applications. Our laboratory has introduced sequence-defined DNA amphiphiles which self-assemble in aqueous solutions. Despite their advantages, self-assembled SNAs can be inherently fragile due to their reliance on non-covalent interactions and fall apart in biologically relevant conditions, specifically by interaction with serum proteins. Herein, this challenge is addressed by introducing two methods of covalent crosslinking of SNAs via UV irradiation. Thymine photodimerization or disulfide crosslinking at the micellar interface enhance SNA stability against human serum albumin binding. This enhanced stability, particularly for disulfide crosslinked SNAs, leads to increased cellular uptake. Furthermore, this crosslinking results in sustained activity and accessibility for release of the therapeutic nucleic acid, along with improvement in unaided gene silencing. The findings demonstrate the efficient stabilization of SNAs through UV crosslinking, influencing their cellular uptake, therapeutic release, and ultimately, gene silencing activity. These studies offer promising avenues for further optimization and exploration of pre-clinical, in vivo studies., (© 2025 The Author(s). Small published by Wiley‐VCH GmbH.)

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. These motifs can be prepared through "DNA printing", which uses a DNA nanostructure as a temporary template to covalently transfer specific DNA strands to a small molecule core. Here, the "printing" process is streamlined with DNA-immobilized polystyrene microspheres, laying the foundation for future automated DNA printing devices. First, the DNA template hybridizes with reactive complementary strands, which are then crosslinked using a small molecule. Second, beads with fully complementary molecules capture the "daughter" products by strand displacement. This ensures high product yields and high recovery of the "mother" template for reuse. This method allows the precise transfer of different DNA strands onto various small molecules, including aromatics and functional porphyrins. Notably, these branching motifs exhibit remarkable stability toward nucleases without any specialized modifications. Moreover, they can serve as robust building blocks for precise assembly of 3D structures, such as an addressable tetrahedron from only two components., (© 2024 The Author(s). Small Methods published by Wiley‐VCH GmbH.)

Nucleic acids, used as therapeutics to silence disease-related genes, offer significant advantages over small molecule drugs: they provide high specificity, the ability to target "undruggable" molecules, and adaptability to a wide range of disease phenotypes. However, their instability in biological media, as well their rapid clearance from the organism limit their applicability, necessitating the use of nanocarriers to overcome these challenges. Among these strategies, spherical nucleic acids (SNA)-composed of a densely packed corona of oligonucleotides around a nanoparticle-have emerged as a powerful tool, in particular when self-assembled from DNA amphiphiles. This non-covalent strategy however has caveats, especially when it comes to stability in complex biological media, where these SNAs disassemble in contact to serum proteins. Here, we developed highly fluorinated DNA amphiphiles that readily self-assemble into SNAs and have tunable stability profiles in biological media. They are made of branched fluorinated moieties with potentially improved biodegradability as compared to their linear counterparts. Depending on the number of fluorophilic interactions, the self-assembled SNAs can have excellent serum stabilities-up to days-and readily deliver nucleic acid therapeutics for gene silencing applications. These systems show great potential as promising candidates for nucleic acid-based therapies., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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. Assembly through base-pairing leads to the formation of hybrid, multi-phasic hydrogels with high stiffness and self-healing properties. Assembly via hydrophobic core interactions gives anisotropic, discrete assemblies, where DNA fibers with one sequence are terminated with DNA spheres bearing different sequences. This work opens new avenues for the bottom-up construction of DNA-based materials, with promising applications in drug delivery, tissue engineering, and the creation of complex DNA structures from a minimum array of components., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

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. Using single-molecule fluorescence imaging, the kinetics and yield of each synthetic step can be quantitatively determined, revealing differences in self-assembly dynamics as the nanotube is built up from the solid support and providing new insights into DNA self-assembly. The exploitation of automation for both assembly and analysis (through an ad-hoc developed K-means clustering algorithm) facilitates a workflow wherein the synthesis parameters may be iteratively improved upon, demonstrating how a single-molecule "assembly-analysis-optimization" sequence can be used to generate complex, noncovalent materials in good yield. The presented synthetic strategy is generalizable, making use of equipment already available in most standard laboratories and represents the first fully automated supramolecular assembly on a solid support., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

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. Here we show that highly charged, monodisperse DNA-oligomers assemble via seeded growth into length-controlled supramolecular fibers during heating; when the temperature is lowered, these metastable fibers slowly disassemble. Furthermore, the specific molecular structures of oligomers that promote fiber formation contradict the typical theory of block copolymer self-assembly. Efficient curling and packing of the oligomers - or 'curlamers' - determine morphology, rather than hydrophobic to hydrophilic ratio. Addition of a small molecule stabilises the DNA fibers, enabling temporal control of polymer lifetime and underscoring their potential use in nucleic-acid delivery, stimuli-responsive biomaterials, and soft robotics., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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. Gene silencing involves multiple steps including cellular uptake, endosomal escape, and therapeutic cargo release. Whether assembly vs disassembly is advantageous to any of these steps has not been previously studied. In this work, we modify the DNA and hydrophobic portions of SaSNAs and examine their effects on stability, cellular uptake, and gene silencing. When the linkages between the hydrophobic units are changed from phosphate to phosphorothioate, we find that the SaSNAs disassemble better in endosomal conditions and exhibit more efficacious silencing, despite having cellular uptake similar to that of their phosphate counterparts. Thus, disassembly in the endolysosomal compartments is advantageous, facilitating the release of the nucleic acid cargo and the interactions between the hydrophobic units and endosomal lipids. We also find that SaSNAs partially disassemble in serum to bind albumin; the disassembled, albumin-bound strands are less efficient at cellular uptake and gene silencing than their assembled counterparts, which can engage scavenger receptors for internalization. When the DNA portion is cross-linked by G-quadruplex formation, disassembly decreases and cellular uptake significantly increases. However, this does not translate to greater gene silencing, again illustrating the need for disassembly of the SaSNAs when they are in the endosome. This work showcases the advantages of the dual nature of SaSNAs for gene silencing, requiring extracellular assembly and disassembly inside the cell compartments.

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.

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., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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. Here, we explore the ability of this novel albumin-binding conjugate to improve the delivery of siRNA in vivo . We demonstrate that the conjugate binds albumin exclusively in circulation and extravasates to various organs, enabling effective gene silencing. Notably, we show that the conjugate achieves a balance between hydrophobicity and safety, as it significantly reduces the side effects associated with siRNA interactions with blood components, which are commonly observed in some hydrophobically conjugated siRNAs. In addition, it reduces siRNA monocyte uptake, which may lead to cytokine/inflammatory responses. This work showcases the potential of using this dendritic conjugate as a selective albumin binding handle for the effective and safe delivery of nucleic acid therapeutics. We envision that these properties may pave the way for new opportunities to overcome delivery hurdles of oligonucleotides in future applications., Competing Interests: H.H.F., A.K., and H.F.S. have filed a patent that covers the use of the dendritic conjugate in vivo for various applications. A.K. discloses ownership of stock in RXi Pharmaceuticals and Advirna; is a founder of Atalanta Therapeutics and Comanche Biopharma; and serves on the scientific advisory boards of Aldena Therapeutics, Prime Medicine, and Alltrna. V.N.H. is an employee of and holds stock options in Comanche Biopharma., (© 2023.)

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., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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. However, to date, no studies have investigated the effect of the hydrophobic polymer sequence on the biological properties of SNAs. In this study, we created a library of ASO conjugates by covalently attaching polymers with linear or branched [dodecanediol phosphate] units and systematically varying polymer sequence and composition. We show that these parameters can significantly impact encapsulation efficiency, gene silencing activity, SNA stability, and cellular uptake, thus outlining optimized polymer architectures for gene silencing.

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., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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 3  Pa, and they lack functionality, limiting their applications. Here, a DNA/small-molecule motif to create stiff hydrogels from unmodified DNA, reaching 10 5  Pa in storage modulus is used. The motif consists of an interaction between polyadenine and cyanuric acid-which has 3-thymine like faces-into multimicrometer supramolecular fibers. The mechanical properties of these hydrogels are readily tuned, they are self-healing and thixotropic. They integrate a high density of small, nontoxic molecules, and are functionalized simply by varying the molecule sidechain. They respond to three independent stimuli, including a small molecule stimulus. These stimuli are used to integrate and release DNA wireframe and DNA origami nanostructures within the hydrogel. The hydrogel is applied as an injectable delivery vector, releasing an antisense oligonucleotide in cells, and increasing its gene silencing efficacy. This work provides tunable, stimuli-responsive, exceptionally stiff all-DNA hydrogels from simple sequences, extending these materials' capabilities., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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 in vivo biodistribution. Herein, we report a systematic investigation, combining experimental and computational design, to modulate the length, flexibility, and longitudinal patterns of wireframe DNA nanotubes. Subtle design changes govern the structure and properties of our nanotubes, which are built from a custom-made, long, and size-defined template strand to which DNA rungs and linkers are attached. Unlike DNA origami, these custom-made strands possess regions with repeating sequences at strategic locations, thereby reducing the number of strands necessary for assembly. Through strand displacement, the nanotubes can be reversibly altered between extended and collapsed morphologies. These design concepts enable fine-tuning of the nanotube stiffness and may pave the way for the development of designer nanotubes for a variety of applications, including the study of cellular internalization, biodistribution, and uptake mechanisms for structures of varied shapes and sizes.

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.

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.

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.

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. When rigidified, our DNA tweezer can be addressed with streptavidin-binding ligands. Full control over the number, separation, and location of these ligands enables site-specific streptavidin recognition; all three arms of the DNA nanotweezer wrap around multiple streptavidin units simultaneously. Our approach combines the simplicity of DNA tile arrays with the size regime normally provided by DNA origami, offering an integrated platform for the use of branched DNA scaffolds as structural building blocks, protein sensors, and dynamic, stimuli-responsive materials., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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. Using single-molecule fluorescence microscopy, we observe that proton dissipation influences the growth mechanism of supramolecular polymerization, healing gaps within fibres and converting highly branched, interwoven networks into nanocable superstructures. Just as the growth kinetics of natural fibres determine their structural attributes to modulate function, our system of photoacid-enabled depolymerization and repolymerization selects for healed materials to yield organized, robust fibres. Our method provides a chemical route for error-checking, distinct from thermal annealing, that improves the morphologies and properties of supramolecular materials using out-of-equilibrium systems., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

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. Enzymatic hydrolysis, low cellular uptake, immune cell recognition and degradation, and unclear biodistribution profiles are yet to be solved. Rigorous methodologies are needed to study, understand, and control the fate of self-assembled DNA structures in physiological conditions. In this review, we describe the current challenges faced by the field as well as recent successes, highlighting the potential to solve biology problems or develop smart drug delivery tools. We then propose an outlook to drive the translation of DNA constructs toward preclinical design. We particularly believe that a detailed understanding of the fate of DNA nanostructures within living organisms, achieved through thorough characterization, is the next required step to reach clinical maturity.

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. Here, we modify SNAs with 2'-deoxy-2'-fluoro-d-arabinonucleic acid (FANA-SNA), and show increased stability, enhanced gene silencing potency and unaided uptake (gymnosis) as compared to free FANA. By varying the spacer region between the nucleic acid strand and the attached hydrophobic polymer, we show that a cleavable DNA based spacer is essential for maximum activity. This design feature will be important when implementing functionalized nucleic acids into nanostructures for gene silencing. The modularity of the FANA-SNA was demonstrated by silencing two different targets. Transfection-free delivery was superior for the modified SNA compared to the free FANA oligonucleotide., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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.

Conjugation of lipid moieties to nucleic-acid therapeutics increases their interaction with cellular membranes, enhances their uptake and influences in vivo distribution. Once injected in biological fluids, such modifications trigger the binding of various serum proteins, which in turn play a major role in determining the fate of oligonucleotides. Yet, the role played by each of these proteins, more than 300 in serum, remains to be elucidated. Albumin, the most abundant circulating protein is an attractive candidate to study, as it was previously used to enhance the therapeutic effect of various drugs. Herein, we present a thorough fluorescent-based methodology to study the effect of strong and specific albumin-binding on the fate and cellular uptake of DNA oligonucleotides. We synthesized a library of molecules that exhibit non-covalent binding to albumin, with affinities ranging from high (nanomolar) to none. Our results revealed that strong albumin binding can be used as a strategy to reduce degradation of oligonucleotides in physiological conditions caused by enzymes (nucleases), to reduce uptake and degradation by immune cells (macrophages) and to prevent non-specific uptake by cells. We believe that introducing protein-binding domains in oligonucleotides can be used as a strategy to control the fate of oligonucleotides in physiological environments. While our study focuses on albumin, we believe that such systematic studies, which elucidate the role of serum proteins systematically, will ultimately provide a toolbox to engineer the next-generation of therapeutic oligonucleotides, overcoming many of the barriers encountered by these therapeutics, such as stability, immunogenicity and off-target effects., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

The construction of metallic nanostructures with customizable morphologies and complex shapes has been an essential pursuit in nanoscience. DNA nanotechnology has enabled the fabrication of increasingly complex DNA nanostructures with unprecedented specificity, programmability and sub-nanometer precision, which makes it an ideal approach to rationally organize metallic nanostructures. Here we report an Assemble, Grow and Lift-Off (AGLO) strategy to construct robust standalone gold nanostructures with pre-designed customizable shapes in solution, using only a simple 2D DNA origami sheet as a versatile transient template. Gold nanoparticle (AuNP) seeds were firstly assembled onto the pre-designed binding sites of the DNA origami template and then additional gold was slowly deposited onto the AuNP seeds. The growing seed surfaces eventually merge with adjacent seeds to generate one continuous gold nanostructure in a pre-designed shape, which can then be lifted off the origami template. Diverse customized patterns of templated AuNP seeds were successfully transformed into corresponding gold nanostructures with the target structure transformation percentage over 80%. Moreover, the AGLO strategy can be incorporated with a magnetic bead separation platform to enable the easy recycling of the excess AuNP seeds and DNA components., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

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 (DX M ) by site-specifically functionalizing the tile crossovers with tetrahedral binding pockets that coordinate Cu I . These DX junctions bind two Cu I 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 Cu II is spontaneously reduced to Cu I in situ. By modifying our design over three generations of tiles, we elucidated the thermodynamic and geometric requirements for the successful assembly of DX M tiles, which have direct applicability in developing robust, stable DNA-based materials with electroactive, photoactive, and catalytic properties., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 Zn 2+ ions, increased their selectivity for Zn 2+ over Co 2+ 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 Zn 2+ ions., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA nanotechnology has been used to create DNA containing nanostructures with well-defined sizes and shapes-properties highly applicable to drug delivery. By appending sequence-defined hydrophobic segments to DNA, DNA amphiphiles are created whose structures and modes of self-assembly mimic specialized biomacromolecules such as proteins. Automated, solid-phase DNA synthesis is a scalable and robust technique that has been optimized for several decades to make DNA oligomers. Using the same method and with minimal additional cost, DNA amphiphiles are synthesized with total control of monomer sequence. A variety of synthetic monomers may be appended to DNA depending on the application, but of particular interest is a linear twelve-carbon alkyl chain (C12). This chapter describes the synthesis, purification, and characterization of a DNA amphiphile consisting of twelve C12 units covalently attached to a 19mer DNA sequence (C12 12 -DNA 19 ). These DNA amphiphiles self-assemble into spherical nanoparticles with potential applications for nucleic acid delivery. Methods common to chemistry and molecular biology are employed, including high-performance liquid chromatography and gel electrophoresis, as well as the more specialized imaging technique of atomic force microscopy.

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.

Fluorescent dye labeling of DNA oligonucleotides and nanostructures is one of the most used techniques to track their fate and cellular localization inside cells. Here, we report that intracellular fluorescence, and even FRET signals, cannot be correlated with the cellular uptake of intact DNA structures. Live cell imaging revealed high colocalization of cyanine-labeled DNA oligos and nanostructures with phosphorylated small-molecule cyanine dyes, one of the degradation products from these DNA compounds. Nuclease degradation of the strands outside and inside the cell results in a misleading intracellular fluorescent signal. The signal is saturated by the fluorescence of the degradation product (phosphorylated dye). To test our hypothesis, we synthesized a range of DNA structures, including Cy3- and Cy5-labeled DNA cubes and DNA tetrahedra, and oligonucleotides with different stabilities toward nucleases. All give fluorescence signals within the mitochondria after cellular uptake and strongly colocalize with a free phosphorylated dye control. Kinetics experiments revealed that uptake of stable DNA structures is delayed. We also studied several parameters influencing fluorescent data: stability of the DNA strand, fixation methods that can wash away the signal, position of the dye on the DNA strand, and design of FRET experiments. DNA nanostructures hold tremendous potential for biomedical applications and biotechnology because of their biocompatibility, programmability, and easy synthesis. However, few examples of successful DNA machines in vivo have been reported. We believe this contribution can be used as a guide to design better cellular uptake experiments when using fluorescent dyes, in order to further propel the biological development, and application of DNA nanostructures., Competing Interests: The authors declare no competing financial interest.

In this work, we report a component-minimal spherical nucleic acid (SNA) from monodisperse DNA-polymer conjugates that can load and release nucleic acid therapeutics in a stimuli-responsive manner. We show that this vehicle assembles from only four strands, and conditional release of its antisense therapeutic cargo can be induced upon recognition of specific oligonucleotide triggers via strand displacement. The latter (triggers) may be a microRNA that offers additional synergistic therapy, in addition to the previously shown ability of the SNA to load hydrophobic drugs. The SNA is easy to prepare, has dynamic character, releases its cargo only upon the presence of both triggers, and can survive biological conditions while protecting its cargo. The gene silencing potency of the cargo was tested in live cells and shown to be suppressed when loaded in the SNA, and its activity was restored only upon release with the two triggers. This vehicle has the essential characteristics of versatility, ease of synthesis, low cost, highly responsive behavior, and ability to support combination therapies, making it a promising candidate for cell-selective drug delivery and clinical transition.

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

DNA nanotechnology enables the design and assembly of DNA nanostructures with unprecedented control over their size and shape. Additionally, the programmable base-pairing alphabet of DNA allows the incorporation of responsive units within these DNA nanostructures. Here, we describe a general design strategy to construct responsive DNA prisms that can encapsulate and selectively release an encapsulated siRNA upon recognition of an oligonucleotide trigger. This prismatic DNA scaffold design is adaptable and can encapsulate oligonucleotides of any length and type. Moreover, the prism can be made to respond to an oligonucleotide trigger of interest like a messenger RNA (mRNA) or a microRNA (miRNA), thus enabling dual or synergistic therapeutic strategies. We present an overview of the design strategy used to access these DNA nanostructures, followed by the steps involved in DNA sequence generation, assembly, and validation of this construct.

Dynamic wireframe DNA structures have gained significant attention in recent years, with research aimed toward using these architectures for sensing and encapsulation applications. For these assemblies to reach their full potential, however, knowledge of the rates of strand displacement and hybridization on these constructs is required. Herein, we report the use of single-molecule fluorescence methodologies to observe the reversible switching between double- and single-stranded forms of triangular wireframe DNA nanotubes. Specifically, by using fluorescently labeled DNA strands, we were able to monitor changes in intensity over time as we introduced different sequences. This allowed us to extract detailed kinetic information on the strand displacement and hybridization processes. Due to the polymeric nanotube structure, the ability to individually address each of the three sides, and the inherent polydispersity of our samples as a result of the step polymerization by which they are formed, a library of compounds could be studied independently yet simultaneously. Kinetic models relying on mono-exponential decays, multi-exponential decays, or sigmoidal behavior were adjusted to the different constructs to retrieve erasing and refilling kinetics. Correlations were made between the kinetic behavior observed, the site accessibility, the nanotube length, and the structural robustness of wireframe DNA nanostructures, including fully single-stranded analogs. Overall, our results reveal how the length, morphology, and rigidity of the DNA framework modulate the kinetics of strand displacement and hybridization as well as the overall addressability and structural stability of the structures under study.

RNA has inherent therapeutic and structural properties that make it an important component of biologically-functional nanoparticles. Using DNA-amphiphiles as synthetic templates, we report the synthesis of two classes of RNA-amphiphiles that self-assemble into spherical nanoparticles in aqueous solution and show gene silencing activity.

DNA is a powerful guiding molecule to achieve the precise construction of arbitrary structures and high-resolution organization of functional materials. The combination of sequence programmability, rigidity and highly specific molecular recognition in this molecule has resulted in a wide range of exquisitely designed DNA frameworks. To date, the impressive potential of DNA nanomaterials has been demonstrated from fundamental research to technological advancements in materials science and biomedicine. This review presents a summary of some of the most recent developments in structural DNA nanotechnology regarding new assembly approaches and efforts in translating DNA nanomaterials into practical use. Recent work on incorporating blunt-end stacking and hydrophobic interactions as orthogonal instruction rules in DNA assembly, and several emerging applications of DNA nanomaterials will also be highlighted., (Copyright © 2018. Published by Elsevier Ltd.)

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.

Understanding how biological macromolecules assemble into higher-order structures is critical to explaining their function in living organisms and engineered biomaterials. Transient, partly-structured intermediates are essential in many assembly processes and pathway selection, but are challenging to characterize. Here we present a simple thermal hysteresis method based on rapid, non-equilibrium melting and annealing measurements that maps the rate of supramolecular assembly as a function of temperature and concentration. A straightforward analysis of these surfaces provides detailed information on the natures of assembly pathways, offering temperature resolution beyond that accessible with conventional techniques. Validating the approach using a tetrameric guanine quadruplex, we obtain strikingly good agreement with previous kinetics measurements and reveal temperature-dependent changes to the assembly pathway. In an application to the recently discovered co-assembly of polydeoxyadenosine (poly(A)) and cyanuric acid, we show that fiber elongation is initiated when an unstable complex containing three poly(A) monomers acquires a fourth strand.

DNA origami is one of the most effective tools for bottom-up construction of novel objects and devices at the nanometer-scale. However, many applications require larger structures than can be obtained with the conventional single-stranded scaffold, typically 7249 nucleotides. Here, we address this limitation by developing custom-made single-stranded scaffolds that bind pre-assembled origami tiles and induce their one-dimensional organization in high yields. Our synthetic method allows the conversion of multiple repetitive and unique sequences into correctly assembled, large backbones, and to finely tune the position and frequency of each building block. Granted with these regions, three and five origami tiles were successfully arranged in 1-D with the aid of one or two scaffolds, forming a nano-"railroad track". This new method increases length scale in DNA origami without increasing cost and complexity, and is anticipated to increase the yield of other approaches aiming to assemble large origami structures.

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.

Natural systems combine different supramolecular interactions in a hierarchical manner to build structures. In contrast, DNA nanotechnology relies almost exclusively on DNA base pairing for structure generation. Introducing other supramolecular interactions can expand the structural and functional range of DNA assemblies, but this requires an understanding of the interplay between these interactions. Here, an economic strategy to build DNA nanotubes functionalized with lipid-like polymers is reported. When these polymers are linked to the nanotube using a spacer, they fold inside to create a hydrophobic environment within the nanotube; the nanotube can encapsulate small molecules and conditionally release them when specific DNA strands are added, as monitored by single-molecule fluorescence microscopy. When the polymers are directly linked to the nanostructure without spacers, they interact intermolecularly to form a network of DNA bundles. This morphological switch can be directly observed using a strand displacement strategy. The two association modes result in different cellular uptake behavior. Nanotubes with internal hydrophobic association show dye-mediated mitochondrial colocalization inside cells; while the bundles disassemble into smaller polymer-coated structures that reduce the extent of nonspecific cellular uptake. This approach uncovers parameters to direct the hierarchical assembly of DNA nanostructures, and produces promising materials for targeted drug delivery., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As colloidal self-assembly increasingly approaches the complexity of natural systems, an ongoing challenge is to generate non-centrosymmetric structures. For example, patchy, Janus or living crystallization particles have significantly advanced the area of polymer assembly. It has remained difficult, however, to devise polymer particles that associate in a directional manner, with controlled valency and recognition motifs. Here, we present a method to transfer DNA patterns from a DNA cage to a polymeric nanoparticle encapsulated inside the cage in three dimensions. The resulting DNA-imprinted particles (DIPs), which are 'moulded' on the inside of the DNA cage, consist of a monodisperse crosslinked polymer core with a predetermined pattern of different DNA strands covalently 'printed' on their exterior, and further assemble with programmability and directionality. The number, orientation and sequence of DNA strands grafted onto the polymeric core can be controlled during the process, and the strands are addressable independently of each other.

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., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A wide variety of approaches have become available for the fabrication of nanomaterials with increasing degrees of complexity, precision, and speed while minimizing cost. Their quantitative characterization, however, remains a challenge. Analytical methods to better inspect and validate the structure and composition of large nanoscale objects are required to optimize their applications in diverse technologies. Here, we describe single-molecule fluorescence-based strategies relying on photobleaching and multiple-color co-localization features toward the characterization of supramolecular structures. By optimizing imaging conditions, including surface passivation, excitation power, frame capture rate, fluorophore choice, buffer media, and antifading agents, we have built a robust method by which to dissect the structure of synthetic nanoscale systems. We showcase the use of our methods by retrieving key structural parameters of four DNA nanotube systems differing in their preparation strategy. Our method rapidly and accurately assesses the outcome of synthetic work building nano- and mesoscale architectures, providing a key tool for product studies in nanomaterial synthesis.