Rational Design of pH‐Responsive DNA Motifs with General Sequence Compatibility

Reconfigurable molecular events are key to molecular machines. In response to external cues, molecular machines rearrange/change their structures to perform certain functions. Such machines exist in nature, for example cell surface receptors, and have been artificially engineered. To be able to build sophisticated and efficient molecular machines for an increasing range of applications, constant efforts have been devoted to developing new mechanisms of controllable structural reconfiguration. Herein, we report a general design principle for pH‐responsive DNA motifs for general DNA sequences (not limited to triplex or i‐motif forming sequences). We have thoroughly characterized such DNA motifs by polyacrylamide gel electrophoresis (PAGE) and fluorescence spectroscopy and demonstrated their applications in dynamic DNA nanotechnology. We expect that it will greatly facilitate the development of DNA nanomachines, biosensing/bioimaging, drug delivery, etc.     

Chemical Regulation of DNA i‐Motifs for Nanobiotechnology and Therapeutics

DNA sequences rich in cytosine have the propensity, under acidic pH, to fold into four‐stranded intercalated DNA structures called i‐motifs. Recent studies have provided significant breakthroughs that demonstrate how chemists can manipulate these structures for nanobiotechnology and therapeutics. The first section of this Minireview discusses the development of advanced functional nanostructures by synthetic conjugation of i‐motifs with organic scaffolds and metal nanoparticles and their role in therapeutics. The second section highlights the therapeutic targeting of i‐motifs with chemical scaffolds and their significance in biology. For this, first we shed light on the long‐lasting debate regarding the stability of i‐motifs under physiological conditions. Next, we present a comparative analysis of recently reported small molecules for specifically targeting i‐motifs over other abundant DNA structures and modulating their function in cellular systems. These advances provide new insights into i‐motif‐targeted regulation of gene expression, telomere maintenance, and therapeutic applications.     

Efficacious Anticancer Drug Delivery Mediated by a pH‐Sensitive Self‐Assembly of a Conserved Tripeptide Derived from Tyrosine Kinase NGF Receptor

We present herein a short tripeptide sequence (Lys–Phe–Gly or KFG) that is situated in the juxtamembrane region of the tyrosine kinase nerve growth factor (Trk NGF) receptors. KFG self‐assembles in water and shows a reversible and concentration‐dependent switching of nanostructures from nanospheres (vesicles) to nanotubes, as evidenced by dynamic light scattering, transmission electron microscopy, and atomic force microscopy. The morphology change was associated with a transition in the secondary structure. The tripeptide vesicles have inner aqueous compartments and are stable at pH 7.4 but rupture rapidly at pH≈6. The pH‐sensitive response of the vesicles was exploited for the delivery of a chemotherapeutic anticancer drug, doxorubicin, which resulted in enhanced cytotoxicity for both drug‐sensitive and drug‐resistant cells. Efficient intracellular release of the drug was confirmed by fluorescence‐activated cell sorting analysis, fluorescence microscopy, and confocal microscopy.     

“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.     

“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.     

Enhancing Biocompatible Stability of DNA Nanostructures Using Dendritic Oligonucleotides and Brick Motifs

The use of DNA‐based nanomaterials in biomedical applications is continuing to grow, yet more emphasis is being put on the need for guaranteed structural stability of DNA nanostructures in physiological conditions. Various methods have been developed to stabilize DNA origami against low concentrations of divalent cations and the presence of nucleases. However, existing strategies typically require the complete encapsulation of nanostructures, which makes accessing the encased DNA strands difficult, or chemical modification, such as covalent crosslinking of DNA strands. We present a stabilization method involving the synthesis of DNA brick nanostructures with dendritic oligonucleotides attached to the outer surface. We find that nanostructures assembled from DNA brick motifs remain stable against denaturation without any chemical modifications. Furthermore, densely coating the outer surface of DNA brick nanostructures with dendritic oligonucleotides prevents nuclease digestion.     

Single‐Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells

DNA is typically impermeable to the plasma membrane due to its polyanionic nature. Interestingly, several different DNA nanostructures can be readily taken up by cells in the absence of transfection agents, which suggests new opportunities for constructing intelligent cargo delivery systems from these biocompatible, nonviral DNA nanocarriers. However, the underlying mechanism of entry of the DNA nanostructures into the cells remains unknown. Herein, we investigated the endocytotic internalization and subsequent transport of tetrahedral DNA nanostructures (TDNs) by mammalian cells through single‐particle tracking. We found that the TDNs were rapidly internalized by a caveolin‐dependent pathway. After endocytosis, the TDNs were transported to the lysosomes in a highly ordered, microtubule‐dependent manner. Although the TDNs retained their structural integrity within cells over long time periods, their localization in the lysosomes precludes their use as effective delivery agents. To modulate the cellular fate of the TDNs, we functionalized them with nuclear localization signals that directed their escape from the lysosomes and entry into the cellular nuclei. This study improves our understanding of the entry into cells and transport pathways of DNA nanostructures, and the results can be used as a basis for designing DNA‐nanostructure‐based drug delivery nanocarriers for targeted therapy.     

A Case Study of the Likes and Dislikes of DNA and RNA in Self‐Assembly

Programmed self‐assembly of nucleic acids (DNA and RNA) is an active research area as it promises a general approach for nanoconstruction. Whereas DNA self‐assembly has been extensively studied, RNA self‐assembly lags much behind. One strategy to boost RNA self‐assembly is to adapt the methods of DNA self‐assembly for RNA self‐assembly because of the chemical and structural similarities of DNA and RNA. However, these two types of molecules are still significantly different. To enable the rational design of RNA self‐assembly, a thorough examination of their likes and dislikes in programmed self‐assembly is needed. The current work begins to address this task. It was found that similar, two‐stranded motifs of RNA and DNA lead to similar, but clearly different nanostructures.     

DNA Nanostructures for Targeted Antimicrobial Delivery

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram‐positive (Bacillus subtilis) and Gram‐negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme‐functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.     

Reversible Reconfiguration of DNA Origami Nanochambers Monitored by Single‐Molecule FRET

Today, DNA nanotechnology is one of the methods of choice to achieve spatiotemporal control of matter at the nanoscale. By combining the peculiar spatial addressability of DNA origami structures with the switchable mechanical movement of small DNA motifs, we constructed reconfigurable DNA nanochambers as dynamic compartmentalization systems. The reversible extension and contraction of the inner cavity of the structures was used to control the distance‐dependent energy transfer between two preloaded fluorophores. Interestingly, single‐molecule FRET studies revealed that the kinetics of the process are strongly affected by the choice of the switchable motifs and/or actuator sequences, thus offering a valid method for fine‐tuning the dynamic properties of large DNA nanostructures. We envisage that the proposed DNA nanochambers may function as model structures for artificial biomimetic compartments and transport systems.     

Designed Spiroketal Protein Modulation

Spiroketals are structural motifs found in many biologically active natural products, which has stimulated considerable efforts toward their synthesis and interest in their use as drug lead compounds. Despite this, the use of spiroketals, and especially bisbenzanulated spiroketals, in a structure‐based drug discovery setting has not been convincingly demonstrated. Herein, we report the rational design of a bisbenzannulated spiroketal that potently binds to the retinoid X receptor (RXR) thereby inducing partial co‐activator recruitment. We solved the crystal structure of the spiroketal–hRXRα–TIF2 ternary complex, and identified a canonical allosteric mechanism as a possible explanation for the partial agonist behavior of our spiroketal. Our co‐crystal structure, the first of a designed spiroketal–protein complex, suggests that spiroketals can be designed to selectively target other nuclear receptor subtypes.     

Fabrication of Defined Polydopamine Nanostructures by DNA Origami‐Templated Polymerization

A versatile, bottom‐up approach allows the controlled fabrication of polydopamine (PD) nanostructures on DNA origami. PD is a biosynthetic polymer that has been investigated as an adhesive and promising surface coating material. However, the control of dopamine polymerization is challenged by the multistage‐mediated reaction mechanism and diverse chemical structures in PD. DNA origami decorated with multiple horseradish peroxidase‐mimicking DNAzyme motifs was used to control the shape and size of PD formation with nanometer resolution. These fabricated PD nanostructures can serve as “supramolecular glue” for controlling DNA origami conformations. Facile liberation of the PD nanostructures from the DNA origami templates has been achieved in acidic medium. This presented DNA origami‐controlled polymerization of a highly crosslinked polymer provides a unique access towards anisotropic PD architectures with distinct shapes that were retained even in the absence of the DNA origami template.     

Precisely Tailored DNA Nanostructures and their Theranostic Applications

A critical challenge in nanotechnology is the limited precision and controllability of the structural parameters, which brings about concerns in uniformity, reproducibility and performance. Self‐assembled DNA nanostructures, as a newly emerged type of nano‐biomaterials, possess low‐nanometer precision, excellent programmability and addressability. They can precisely arrange various molecules and materials to form spatially ordered complex, resulting in unambiguous physical or chemical properties. Because of these, DNA nanostructures have shown great promise in numerous biomedical theranostic applications. In this account, we briefly review the history and advances on construction of DNA nanoarchitectures and superstructures with accurate structural parameters. We focus on recent progress in exploiting these DNA nanostructures as platforms for quantitative biosensing, intracellular diagnosis, imaging, and smart drug delivery. We also discuss key challenges in practical applications.     

One‐Step Formation of “Chain‐Armor”‐Stabilized DNA Nanostructures

DNA‐based self‐assembled nanostructures are widely used to position organic and inorganic objects with nanoscale precision. A particular promising application of DNA structures is their usage as programmable carrier systems for targeted drug delivery. To provide DNA‐based templates that are robust against degradation at elevated temperatures, low ion concentrations, adverse pH conditions, and DNases, we built 6‐helix DNA tile tubes consisting of 24 oligonucleotides carrying alkyne groups on their 3′‐ends and azides on their 5′‐ends. By a mild click reaction, the two ends of selected oligonucleotides were covalently connected to form rings and interlocked DNA single strands, so‐called DNA catenanes. Strikingly, the structures stayed topologically intact in pure water and even after precipitation from EtOH. The structures even withstood a temperature of 95 °C when all of the 24 strands were chemically interlocked.     

Self‐Assembly of Hierarchical Nanostructures from Dopamine and Polyoxometalate for Oral Drug Delivery

The preparation of 3D hierarchical nanostructures by a simple and versatile strategy of self‐assembly of dopamine (DA) and phosphotungstic acid (PTA) is described. The size and morphology of the hierarchical nanostructures could be simply controlled by varying the ratio of the two components, their concentrations, and the pH of the initial Tris‐HCl solution. The self‐assembly of the flowerlike microspheres has been found to involve a two‐stage growth process. Moreover, use of the hierarchical nanostructures as a possible carrier for an anticancer drug in chemotherapy has been explored. The nanostructures showed an intriguing pH‐dependent release behavior, making them promising for applications in biomedical science.     

DNA-based machines

Nucleic acids include substantial information in their base sequence and their hybridization-complexation motifs. Recent research efforts attempt to utilize this biomolecular information to develop DNA nanostructures exhibiting machine-like functions. DNA nano-assemblies revealing tweezers, motor, and walker activities exemplify a few such machines. The DNA-based machines provide new components that act as sensitive sensors, transporters, or drug delivery systems.     

Triplex DNA Nanostructures: From Basic Properties to Applications

Triplex nucleic acids have recently attracted interest as part of the rich “toolbox” of structures used to develop DNA‐based nanostructures and materials. This Review addresses the use of DNA triplexes to assemble sensing platforms and molecular switches. Furthermore, the pH‐induced, switchable assembly and dissociation of triplex‐DNA‐bridged nanostructures are presented. Specifically, the aggregation/deaggregation of nanoparticles, the reversible oligomerization of origami tiles and DNA circles, and the use of triplex DNA structures as functional units for the assembly of pH‐responsive systems and materials are described. Examples include semiconductor‐loaded DNA‐stabilized microcapsules, DNA‐functionalized dye‐loaded metal–organic frameworks (MOFs), and the pH‐induced release of the loads. Furthermore, the design of stimuli‐responsive DNA‐based hydrogels undergoing reversible pH‐induced hydrogel‐to‐solution transitions using triplex nucleic acids is introduced, and the use of triplex DNA to assemble shape‐memory hydrogels is discussed. An outlook for possible future applications of triplex nucleic acids is also provided.     

An Unprecedented Knot‐like G‐Quadruplex Peripheral Motif

A knot‐like G‐quadruplex peripheral structure is formed by a 7‐nt DNA sequence DL7 (TGTTGGT), in which six out of its seven nucleobases participate in compact base‐pairing interactions. Here, the solution NMR structure of a 24‐nt DNA oligonucleotide containing the DL7 sequence shows the interaction between a two‐layer anti‐parallel G‐quadruplex core and the peripheral knot‐like structure, including the construction of two sharp turns in the DNA backbone. The formation of this novel structural element highlights the intricate properties of single‐stranded DNA folding in presence of G‐quadruplex‐forming motifs. We demonstrated the compatibility of the DL7 knot‐like structure with various G‐quadruplexes, which could have implications in drug design and DNA engineering.     

Cluster‐based molecular docking study for in silico identification of novel 6‐fluoroquinolones as potential inhibitors against Mycobacterium tuberculosis

A classical protein sequence alignment and homology modeling strategy were used for building three Mycobacterium tuberculosis‐DNA gyrase protein models using the available topoII‐DNA‐6FQ crystal structure complexes originating from different organisms. The recently determined M. tuberculosis‐DNA gyrase apoprotein structures and topoII‐DNA‐6FQ complexes were used for defining the 6‐fluoroquinolones (6‐FQs) binding pockets. The quality of the generated models was initially validated by docking of the cocrystallized ligands into their binding site, and subsequently by quantitative evaluation of their discriminatory performances (identification of active/inactive 6‐FQs) for a set of 145 6‐FQs with known biological activity values. The M. tuberculosis‐DNA gyrase model with the highest estimated discriminatory power was selected and used afterwards in an additional molecular docking experiment on a mixed combinatorial set of 427 drug‐like 6‐FQ analogs for which the biological activity values were predicted using a prebuilt counter‐propagation artificial neural network model. A novel three‐level Boolean‐based [T/F (true/false)] clustering algorithm was used to assess the generated binding poses: Level 1 (geometry properties assessment), Level 2 (score‐based clustering and selection of the (T)‐signed highly scored Level 1 poses), and Level 3 (activity‐based clustering and selection of the most “active” (T)‐signed Level 2 hits). The frequency analysis of occurrence of the fragments attached at R₁ and R₇ position of the (T)‐signed 6‐FQs selected in Level 3 revealed several novel attractive fragments and confirmed some previous findings. We believe that this methodology could be successfully used in establishing novel possible structure‐activity relationship recommendations in the 6‐FQs optimization, which could be of great importance in the current antimycobacterial hit‐to‐lead processes. © 2012 Wiley Periodicals, Inc.