Investigation of Genetic Association Between a High Activity Variant of Cathepsin G and Risk for Basal Cell Carcinoma.

BACKGROUND/AIM: Cathepsin G (CTSG) has been identified as an inhibitor of breast, bladder, and colorectal cancers. The G allele of the N125S (A/G, rs45567233) functional polymorphism of the CTSG gene confers increased serum CTSG activity and has been associated with cardiovascular and neurovascular diseases. This study examined the possible correlation between the pathogenesis of basal cell carcinoma (BCC) and the functional polymorphism CTSG N125S. PATIENTS AND METHODS: A total of 197 DNA samples were examined, comprising 98 BCC patients and 99 control samples of Greek origin. The CTSG N125S polymorphism was molecularly genotyped using PCR amplification, followed by enzyme digestion, and agarose gel electrophoresis of the amplified DNA fragments. RESULTS: There was no statistically significant difference in the genotypic and allelic frequencies between the patient and the control groups. CONCLUSION: There is no association between the CTSG N125S polymorphism and pathogenesis of BCC.

Advances in DNA-based electrochemical biosensors for the detection of foodborne pathogenic bacteria.

The detection of foodborne pathogenic bacteria is critical in preventing foodborne diseases. DNA-based electrochemical biosensors, with the merits of high sensitivity and short detection time, provide an effective detecting method for foodborne pathogens, attracting significant interest for the past few years. This review mainly describes the important research progress of DNA-based electrochemical biosensors for the detection of foodborne pathogenic bacteria through four perspectives: representative foodborne pathogens detection using electrochemical approaches, DNA immobilization strategies of aptamers, DNA-based signal amplification strategies used in electrochemical DNA sensors, and functional DNA used in electrochemical DNA sensors. Finally, perspectives and challenges are presented in this field. This review will contribute to DNA-based electrochemical biosensor in enhancing the nucleic acid signal amplification.

Rationally Designed DNA-Based Scaffolds and Switching Probes for Protein Sensing.

The detection of a protein analyte and use of this type of information for disease diagnosis and physiological monitoring requires methods with high sensitivity and specificity that have to be also easy to use, rapid and, ideally, single step. In the last 10 years, a number of DNA-based sensing methods and sensors have been developed in order to achieve quantitative readout of protein biomarkers. Inspired by the speed, specificity, and versatility of naturally occurring chemosensors based on structure-switching biomolecules, significant efforts have been done to reproduce these mechanisms into the fabrication of artificial biosensors for protein detection. As an alternative, in scaffold DNA biosensors, different recognition elements (e.g., peptides, proteins, small molecules, and antibodies) can be conjugated to the DNA scaffold with high accuracy and precision in order to specifically interact with the target protein with high affinity and specificity. They have several advantages and potential, especially because the transduction signal can be drastically enhanced. Our aim here is to provide an overview of the best examples of structure switching-based and scaffold DNA sensors, as well as to introduce the reader to the rational design of innovative sensing mechanisms and strategies based on programmable functional DNA systems for protein detection.

Advancing In Situ Food Monitoring through a Smart Lab-in-a-Package System Demonstrated by the Detection of Salmonella in Whole Chicken.

With food production shifting away from traditional farm-to-table approaches to efficient multistep supply chains, the incidence of food contamination has increased. Consequently, pathogen testing via inefficient culture-based methods has increased, despite its lack of real-time capabilities and need for centralized facilities. While in situ pathogen detection would address these limitations and enable individual product monitoring, accurate detection within unprocessed, packaged food products without user manipulation has proven elusive. Herein, "Lab-in-a-Package" is presented, a platform capable of sampling, concentrating, and detecting target pathogens within closed food packaging, without intervention. This system consists of a newly designed packaging tray and reagent-infused membrane that can be paired universally with diverse pathogen sensors. The inclined food packaging tray maximizes fluid localization onto the sensing interface, while the membrane acts as a reagent-immobilizing matrix and an antifouling barrier for the sensor. The platform is substantiated using a newly discovered Salmonella-responsive nucleic acid probe, which enables hands-free detection of 103 colony forming units (CFU) g-1 target pathogen in a packaged whole chicken. The platform remains effective when contamination is introduced with toolsand surfaces, ensuring widespread efficacy. Its real-world use for in situ detection is simulated using a handheld fluorescence scanner with smartphone connectivity.

Functional DNA sensors integrated with nucleic acid signal amplification strategies for non-nucleic acid targets detection.

In addition to carrying and transmitting genetic material, some DNA molecules have specific binding ability or catalytic function. DNA with this special function is collectively referred to as functional DNA (fDNA), such as aptamer, DNAzyme and so on. fDNA has the advantages of simple synthetic process, low cost and low toxicity. It also has high chemical stability, recognition specificity and biocompatibility. In recent years, fDNA biosensors have been widely investigated as signal recognition elements and signal transduction elements for the detection of non-nucleic acid targets. However, the main problem of fDNA sensors is their limited sensitivity to trace targets, especially when the affinity of fDNA to the targets is low. To further improve the sensitivity, various nucleic acid signal amplification strategies (NASAS) are explored to improve the limit of detection of fDNA. In this review, we will introduce four NASAS (hybridization chain reaction, entropy-driven catalysis, rolling circle amplification, CRISPR/Cas system) and the corresponding design principles. The principle and application of these fDNA sensors integrated with signal amplification strategies for detection of non-nucleic acid targets are summarized. Finally, the main challenges and application prospects of NASAS integrated fDNA biosensing system are discussed.

Dual-signal output fluorescent aptasensor based on DNA programmability and gold nanoflowers for multiple mycotoxins detection.

Herein, a dual-signal output fluorescent aptamer sensor was constructed for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA) using the specific recognition ability of aptamers and the programmability of DNA. A functional capture probe (cDNA) was designed with the black hole quenching motif BHQ1 labeled at the 5' end and biotin (bio) labeled at the 3' end. The fluorescent dye Cy3-labeled aflatoxin B1 aptamer (AFB1-Apt) and the carboxyfluorescein FAM-labeled ochratoxin A aptamer (OTA-Apt) were used as two fluorescent probes. The cDNA is anchored to the quenching material gold nanoflowers (AuNFs) by the action of streptavidin (SA) and biotin. Its ends can be complementarily paired with two fluorescent probe bases to form a double-stranded structure. The fluorescence of Cy3 was quenched by AuNFs, and the fluorescence of FAM was quenched by BHQ1 through the fluorescence energy resonance transfer (FRET) effect, forming a fluorescence quenching system. Due to the high affinity of the target and the aptamer, the structure of the aptamer probe changes and detaches from the sensor when AFB1 and OTA are present, resulting in enhanced fluorescence. Under optimal conditions, the linear range of AFB1 was 0.1-100 ng/mL (R2 = 0.996), the limit of detection (LOD) was as low as 0.014 ng/mL, and the limit of quantification (LOQ) was 0.046 ng/mL. The linear range of OTA was 0.1-100 ng/mL (R2 = 0.995), the limit of detection (LOD) was as low as 0.027 ng/mL, and the limit of quantification (LOQ) was 0.089 ng/mL. The sensor had high accuracy in detecting both AFB1 and OTA in real sample analysis. The results of the t test show that there is no significant difference between the results of this study and the high-performance liquid phase (HPLC) method, indicating that the prepared sensor can be used as a potential platform for multiple mycotoxins detection.

Chemistries and applications of DNA-natural product conjugate.

Natural products and their derivatives have made great contributions to chemotherapy, especially for the treatment of tumors and infections. Despite the achievements, natural product-based small molecule drugs usually suffer from side effects, short circulation time, and solubility issue. To overcome these drawbacks, a common approach is to integrate another bio-functional motif into a natural product compound, enabling targeted or synergistic therapy. One of the most promising strategies is to form a DNA-natural product conjugate to improve therapeutic purposes. The incorporated DNA molecules can serve as an aptamer, a nucleic-acid-based congener of antibody, to specifically bind to the disease target of interest, or function as a gene therapy agent, such as immuno-adjuvant or antisense, to enable synergistic chemo-gene therapy. DNA-natural product conjugate can also be incorporated into other DNA nanostructures to improve the administration and delivery of drugs. This minireview aims to provide the chemistry community with a brief overview on this emerging topic of DNA-natural product conjugates for advanced therapeutics. The basic concepts to use the conjugation, the commonly used robust conjugation chemistries, as well as applications in targeted therapy and synergistic therapy of using DNA-natural product conjugates, are highlighted in this minireview. Future perspectives and challenges of this field are also discussed in the discussion and perspective section.

A Biomimetic Approach for Spatially Controlled Cell Membrane Engineering Using Fusogenic Spherical Nucleic Acid.

Engineering of the cell plasma membrane using functional DNA is important for studying and controlling cellular behaviors. However, most efforts to apply artificial DNA interactions on cells are limited to external membrane surface due to the lack of suitable synthetic tools to engineer the intracellular side, which impedes many applications in cell biology. Inspired by the natural extracellular vesicle-cell fusion process, we have developed a fusogenic spherical nucleic acid construct to realize robust DNA functionalization on both external and internal cell surfaces via liposome fusion-based transport (LiFT) strategy, which enables applications including the construction of heterotypic cell assembly for programmed signaling pathway and detection of intracellular metabolites. This approach can engineer cell membranes in a highly efficient and spatially controlled manner, allowing one to build anisotropic membrane structures with two orthogonal DNA functionalities.

Expression of Two Rye CENH3 Variants and Their Loading into Centromeres.

Gene duplication and the preservation of both copies during evolution is an intriguing evolutionary phenomenon. Their preservation is related to the function they perform. The central component of centromere specification and function is the centromere-specific histone H3 (CENH3). Some cereal species (maize, rice) have one copy of the gene encoding this protein, while some (wheat, barley, rye) have two. Therefore, they represent a good model for a comparative study of the functional activity of the duplicated CENH3 genes and their protein products. We determined the organization of the CENH3 locus in rye (Secale cereale L.) and identified the functional motifs in the vicinity of the CENH3 genes. We compared the expression of these genes at different stages of plant development and the loading of their products, the CENH3 proteins, into nucleosomes during mitosis and meiosis. Using extended chromatin fibers, we revealed patterns of loading CENH3 proteinsinto polynucleosomal domains in centromeric chromatin. Our results indicate no sign of neofunctionalization, subfunctionalization or specialization in the gene copies. The influence of negative selection on the coding part of the genes led them to preserve their conserved function. The advantage of having two functional genes appears as the gene-dosage effect.

Amplified electrochemical biosensing based on bienzymatic cascade catalysis confined in a functional DNA structure.

Herein, an amplified and renewable electrochemical biosensor was developed via bienzymatic cascade catalysis of glucose oxidase (GOx) and horseradish peroxidase (HRP), which were confined in a functional Y-shaped DNA nanostructure oriented by a dual-thiol-ended hairpin probe (dSH-HP) with a paired stem as a rigid scaffold and unpaired loop as enclosed binding platform. For proof-of-concept assay of sequence-specific biomarker DNA related to Alzheimer's disease (aDNA), GOx and redox ferrocene-modified HRP (Fc@HRP) were chemically conjugated in two enzyme strands (GOx-ES1 and Fc@HRP-ES2), respectively. The repeated recycling of aDNA was powered by the displacement of GOx-ES1 by aDNA and exonuclease III (ExoIII)-assisted cleavage reaction for amplified output of numerous GOx-ES1 as dependent transducers, together with Fc@HRP-ES2 which was simultaneously hybridized with dSH-HP to assemble this DNA structure. Rationally, the bienzymatic cascade catalysis was motivated through GOx-catalyzed glucose oxidization to in situ generate hydrogen peroxide (H2O2) and overlapped HRP-catalyzed H2O2 decomposition to promote the electron transfer, producing significantly enhanced electrochemical signal of Fc with an ultrahigh sensitivity down to 0.22 fM of aDNA. Benefited from the unique design of dSH-HP-oriented bienzymatic cascades, this one-step strategy without non-specific blockers passivation was simple and renewable, and would pave a promising avenue for sensitive electrochemical assay of biomolecules.

Aptamer-Integrated Scaffolds for Biologically Functional DNA Origami Structures.

The manufacture of DNA origami nanostructures with highly ordered functional motifs is of great significance for biomedical applications. Here, we present a robust strategy to produce customized scaffolds with integrated aptamer sequences, which enables direct construction of functional DNA origami structures. As we demonstrated, aptamers of various numbers and types were efficiently and stably integrated in user-defined positions of the scaffolds. Specifically, two different thrombin aptamer sequences were simultaneously inserted into the M13mp18 phage genome. The assembled functional DNA origami structures from this aptamer-integrated scaffold exhibited increased binding efficiency to thrombin and displayed more than 10-fold stronger resistance to exonuclease degradation than that produced using the traditional staple extension method. Additionally, a scaffold integrated with the platelet-derived growth factor aptamer was produced, and the assembled DNA origami structures showed significant inhibitory effect on breast cancer cells MDA-MB-231. This scalable method of creating design-specific scaffolds opens up a new way to construct more stable and functionally robust DNA origami structures and thus provides an important basis for their broader applications.

Metal enhanced chemiluminescence nanosensor for ultrasensitive bioassay based on silver nanoparticles modified functional DNA dendrimer.

A novel metal enhanced chemiluminescence (MEC) nanosensor was developed for ultrasensitive biosensing and imaging, based on functional DNA dendrimer (FDD), proximity-dependent DNAzyme and silver nanoparticles (AgNPs). The FDD containing two split G-quadruplex structures was prepared through an enzyme-free and step-by-step assembly strategy, and then reacted with AgNPs and hemin molecules to form the FDD/hemin/AgNPs facilely. Such a MEC nanosensor consisted of three modules: FDD (scaffold), the generated G-quadruplex/hemin DNAzyme (signal reporter) and AgNPs (chemiluminescence enhancer). The MEC effect was achieved by controlling the length of DNA sequences between AgNPs on the periphery of FDD and DNAzymes inside it. Such nanosensor exhibited 9-fold amplification and another 6.4-fold metal enhancement in chemiluminescence intensity, which can be easily applied into trace detection of multiple protein markers using a disposable protein immunoarray. The FDD/hemin/AgNPs-based multiplex MEC imaging assay showed wide linear ranges over 5 orders of magnitude and detection limits down to 5× 10-5 ng L-1 and 1.8 × 10-4 U mL-1 for cardiac troponin T and carcinoma antigen 125, demonstrating a promising potential in application to protein analysis and clinical diagnosis. Moreover, the MEC nanosensor can be effectively delivered into cells with excellent biocompatibility and outstanding stability, offering a new tool for detection of intracellular targets and suggesting wide applications in bioassay.

Holographic Optical Tweezers and Boosting Upconversion Luminescent Resonance Energy Transfer Combined Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a Biosensors.

Taking advantage of outstanding precision in target recognition and trans-cleavage ability, the recently discovered CRISPR/Cas12a system provides an alternative opportunity for designing fluorescence biosensors. To fully exploit the analytical potential, we introduce here some meaningful concepts. First, the collateral cleavage of CRISPR/Cas12a is efficiently activated in a functional DNA regulation manner and the bottleneck which largely applicable to nucleic acids detection is broken. After selection of a representative aptamer and DNAzyme as the transduction pathways, the sensing coverage is extended to a small organic compound (ATP) and a metal ion (Na+). The assay sensitivity is significantly improved by utilizing a bead-supported enrichment strategy wherein emerging holographic optical tweezers are used to enhance imaging stability and simultaneously achieve multiflux analysis. Last, a sandwich-structured energy-concentrating upconversion nanoparticle triggered boosting luminescent resonance energy transfer mode is comined to face with complicated biological samples by skillfully confining the emitters into a very limited inner shell. Following the above attempts, the developed CRISPR/Cas12a biosensors not only present an ultrasensitive assay behavior toward these model non-nucleic acid analytes but also can serve as a formidable toolbox for determining real samples including single cell lysates and human plasma, proving a good practical application capacity.

A CTCF-Binding Element and Histone Deacetylation Cooperatively Maintain Chromatin Loops, Linking to Long-Range Gene Regulation in Cancer Genomes.

BACKGROUND: Genes spanning long chromosomal domains are coordinately regulated in human genome, which contribute to global gene dysregulation and carcinogenesis in cancer. It has been noticed that epigenetic modification and chromatin architecture may participate in the regulation process. However, the regulation patterns and functional elements of long-range gene regulation are unclear. METHODS: Based on the clinical transcriptome data from different tumor sets, a novel expressional correlation analysis pipeline was performed to classify the co-regulated regions and subsets of intercorrelated regions. The GLAM2 program was used to predict conserved DNA elements that enriched in regions. Two conserved elements were selected to delete in Ishikawa and HeLa cells by CRISPR-Cas9. SAHA treatment and HDAC knockdown were used to change the histone acetylation status. Using qPCR, MTT, and scratch healing assay, we evaluate the effect on gene expression and cancer cell phenotype. By DNA pull-down and ChIP, the element-binding proteins were testified. 3C and 3D-FISH were performed to depict the alteration in chromatin architecture. RESULTS: In multiple cancer genomes, we classified subsets of coordinately regulated regions (sub-CRRs) that possibly shared the same regulatory mechanisms and exhibited similar expression patterns. A new conserved DNA element (CRE30) was enriched in sub-CRRs and associated with cancer patient survival. CRE30 could restrict gene regulation in sub-CRRs and affect cancer cell phenotypes. DNA pull-down showed that multiple proteins including CTCF were recruited on the CRE30 locus, and ChIP assay confirmed the CTCF-binding signals. Subsequent results uncovered that as an essential element, CRE30 maintained chromatin loops and mediated a compact chromatin architecture. Moreover, we found that blocking global histone deacetylation induced chromatin loop disruption and CTCF dropping in the region containing CRE30, linked to promoted gene regulation. Additionally, similar effects were observed with CRE30 deletion in another locus of chromosome 8. CONCLUSIONS: Our research clarified a new functional element that recruits CTCF and collaborates with histone deacetylation to maintain high-order chromatin organizations, linking to long-range gene regulation in cancer genomes. The findings highlight a close relationship among conserved DNA element, epigenetic modification, and chromatin architecture in long-range gene regulation process.

Public data sources for regulatory genomic features.

High-throughput technologies have led to a continuously growing amount of information about regulatory features in the genome. A wealth of data generated by large international research consortia is available from online databases. Disease-driven studies provide details on specific DNA elements or epigenetic modifications regulating gene expression in specific cellular and developmental contexts, but these results are usually only published in scientific articles. All this information can be helpful in interpreting variants in the regulatory genome. This review describes a selection of high-profile data sources providing information on the non-coding genome, as well as pitfalls and techniques to search and capture information from the literature.

The Recent Development of Hybridization Chain Reaction Strategies in Biosensors.

With the continuous development of biosensors, researchers have focused increasing attention on various signal amplification strategies to pursue superior performance for more applications. In comparison with other signal amplification strategies, hybridization chain reaction (HCR) as a powerful signal amplification technique shows its certain charm owing to nonenzymatic and isothermal features. Recently, on the basis of conventional HCR, this technique has been developed and improved rapidly, and a variety of HCR-based biosensors with excellent performance have been reported. Herein, we present a systematic and critical review on the research progress of HCR in biosensors in the last five years, including the newly developed HCR strategies such as multibranched HCR, migration HCR, localized HCR, in situ HCR, netlike HCR, and so on, as well as the combination strategies of HCR with isothermal signal amplification techniques, nanomaterials, and functional DNA molecules. By illustrating some representative works, we also summarize the advantage and challenge of HCR in biosensors, and offer a deep discussion of the latest progress and future development trends of HCR in biosensors.

A boosting upconversion luminescent resonance energy transfer and biomimetic periodic chip integrated CRISPR/Cas12a biosensor for functional DNA regulated transduction of non-nucleic acid targets.

Apart from gene editing capacity, the newly discovered CRISPR/Cas systems offer an exciting option for biosensing field because of their excellent target recognition accuracy. However, the currently constructed sensors are not only limited to nucleic acid analysis but also suffer from poor adaptability in complex samples and unsatisfying sensitivity. We herein introduce some advanced concepts to break through these bottlenecks. First, the sensing targets are extended by skillfully designing a functional DNA such as aptamer (for protein) and DNAzyme (for metal ion) to regulate the transduction of non-nucleic acid species and further activate the trans cleavage of CRISPR/Cas12a. Second, a boosting upconversion luminescent resonance energy is triggered by using a peculiar energy-confining notion, whereby the luminescence domain is intensively restricted in a very narrow space (~2.44 nm) and up to 92.9% of the green emission can be quenched by the approaching BHQ-1 modified reporters. Third, a bio-inspired periodic arrangement biomimetic chip (photonic crystal) is employed to selectively reflect the upconversion luminescence to achieve noteworthy signal enhancement (~35-fold). By utilizing very simple detection devices (a 980 nm portable laser and a smartphone), the CRISPR/Cas12a biosensor shows commendable sensitivity and specificity toward model targets (ATP and Na+, limits of detection are ~ 18 nM and ~0.37 µM, respectively). More importantly, the analysis of real complex samples demonstrate that the as-proposed platform can work as a powerful toolbox for monitoring the ATP fluctuation in single cell and point-of-care testing Na+ in human plasma, enabling a broad application prospect.

Fabrication of electrochemical biosensor composed of multi-functional DNA/rhodium nanoplate heterolayer for thyroxine detection in clinical sample.

Thyroxine (T4) contains four iodine atoms and is a major thyroid hormone synthesized in the thyroid gland. Abnormal levels of T4 in the body cause various endocrine diseases. The present study describes the fabrication of an electrochemical biosensor composed of a multi-functional DNA structure/rhodium nanoplates heterolayer for precise detection of T4 concentration. A DNA 3-way junction (3WJ) structure was designed as a multi-functional bioprobe to perform several functions (including target detection, electrochemical signal reporting, and immobilization) simultaneously. Binding between T4 and the T4 DNA aptamer was confirmed through enzyme-linked aptamer assays (ELAAs) and filtration experiments. The multi-functional DNA was immobilized on porous rhodium nanoplates (pRhNPs)-heterolayer modified Au micro-gap electrode. The pRhNPs provided an increment in the surface area and amplification of the electrochemical signal. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to detect T4. Under optimal conditions, the limit of detection of T4 was found to be 10.33 pM. Furthermore, up to 11.41 pM of T4 could be detected in clinical samples. This study demonstrates the possibility of label-free detection of the T4 with multi-functional DNA/pRhNPs heterolayer that can be applied to small molecule detection platform in the near future.

Engineering Functional DNA-Protein Conjugates for Biosensing, Biomedical, and Nanoassembly Applications.

DNA and protein are the most important two classes of biomacromolecules forming the basis of life. The conjugation of the two using crosslinking chemistries enables a combination of molecular recognition, enzymatic catalysis, and Watson-Crick hybridization properties. The DNA-protein conjugate with combined properties enables a broad range of applications, such as sensitive and selective bioassays, therapeutic agents, and building blocks for programmable nanoassemblies. In this review, we survey the conjugates from the aspects of conjugation chemistries as well as applications in biomedical and nanotechnology fields. We highlight the functions of both biological moieties of a conjugate for target binding and signal transduction in bioassays. We also review the use of DNA-protein conjugates for the construction of a variety of functional and dynamic nanostructures, from isolated hybrid cages to three-dimensional (3D) protein crystalline lattices. Moreover, these conjugates have been used as carriers to deliver enzymes or functional nucleic acids for disease treatments and gene editing.

Molecular architectonics of DNA for functional nanoarchitectures.

DNA is the key biomolecule central to almost all processes in living organisms. The eccentric idea of utilizing DNA as a material building block in molecular and structural engineering led to the creation of numerous molecular-assembly systems and materials at the nanoscale. The molecular structure of DNA is believed to have evolved over billions of years, with structure and stability optimizations that allow life forms to sustain through the storage and transmission of genetic information with fidelity. The nanoscale structural characteristics of DNA (2 nm thickness and ca. 40-50 nm persistence length) have inspired the creation of numerous functional patterns and architectures through noncovalent conventional and unconventional base pairings as well as through mutual templating-interactions with small organic molecules and metal ions. The recent advancements in structural DNA nanotechnology allowed researchers to design new DNA-based functional materials with chemical and biological properties distinct from their parent components. The modulation of structural and functional properties of hybrid DNA ensembles of small functional molecules (SFMs) and short oligonucleotides by adapting the principles of molecular architectonics enabled the creation of novel DNA nanoarchitectures with potential applications, which has been termed as templated DNA nanotechnology or functional DNA nanoarchitectonics. This review highlights the molecular architectonics-guided design principles and applications of the derived DNA nanoarchitectures. The advantages and ability of functional DNA nanoarchitectonics to overcome the trivial drawbacks of classical DNA nanotechnology to fulfill realistic and practical applications are highlighted, and an outlook on future developments is presented.