DNA Transformations for Diagnosis and Therapy.

Due to its unique physical and chemical characteristics, DNA, which is known only as genetic information, has been identified and utilized as a new material at an astonishing rate. The role of DNA has increased dramatically with the advent of various DNA derivatives such as DNA-RNA, DNA-metal hybrids, and PNA, which can be organized into 2D or 3D structures by exploiting their complementary recognition. Due to its intrinsic biocompatibility, self-assembly, tunable immunogenicity, structural programmability, long stability, and electron-rich nature, DNA has generated major interest in electronic and catalytic applications. Based on its advantages, DNA and its derivatives are utilized in several fields where the traditional methodologies are ineffective. Here, the present challenges and opportunities of DNA transformations are demonstrated, especially in biomedical applications that include diagnosis and therapy. Natural DNAs previously utilized and transformed into patterns are not found in nature due to lack of multiplexing, resulting in low sensitivity and high error frequency in multi-targeted therapeutics. More recently, new platforms have advanced the diagnostic ability and therapeutic efficacy of DNA in biomedicine. There is confidence that DNA will play a strong role in next-generation clinical technology and can be used in multifaceted applications.

Short RNA Universal Coding for Topological Transformation Nano-barcoding Application.

With the advent of innovative genomic discovery toolkits such as RT-PCR, genetic information can be quickly decrypted, and this has resulted in significant progress in overcoming diseases. However, RT-PCR has the serious problem of frequent errors, and the demand for a new gene diagnostic system is emerging. Herein, we propose a universal coding system for the effective detection of short single-stranded DNA or RNA by using a topological transformation-based nano-barcoding technique (TNT). Our goal was to develop a dedicated diagnostic device that unifies the other gene groups, thus resulting in minimum testing. In a universal coding system consisting of two separate circulation structures, different gene groups become generalized into specific single genes with the same sequence by a strand-displacement reaction and are then amplified, eventually being quickly detected in one TNT system. Simple gene diagnostic systems like this make high-speed, point-of-care diagnostic technologies, and we are very confident that these will provide clinical gene detection in the near future.

Anti-senescence ion-delivering nanocarrier for recovering therapeutic properties of long-term-cultured human adipose-derived stem cells.

BACKGROUND: Human adipose-derived stem cells (hADSCs) have been used in various fields of tissue engineering because of their promising therapeutic efficacy. However, the stemness of hADSCs cannot be maintained for long durations, and their therapeutic cellular functions, such as paracrine factor secretion decrease during long-term cell culture. To facilitate the use of long-term-cultured hADSCs (L-ADSCs), we designed a novel therapeutic anti-senescence ion-delivering nanocarrier (AIN) that is capable of recovering the therapeutic properties of L-ADSCs. In the present study, we introduced a low-pH-responsive ion nanocarrier capable of delivering transition metal ions that can enhance angiogenic paracrine factor secretion from L-ADSCs. The AINs were delivered to L-ADSCs in an intracellular manner through endocytosis. RESULTS: Low pH conditions within the endosomes induced the release of transition metal ions (Fe) into the L-ADSCs that in turn caused a mild elevation in the levels of reactive oxygen species (ROS). This mild elevation in ROS levels induced a downregulation of senescence-related gene expression and an upregulation of stemness-related gene expression. The angiogenic paracrine factor secretion from L-ADSCs was significantly enhanced, and this was evidenced by the observed therapeutic efficacy in response to treatment of a wound-closing mouse model with conditioned medium obtained from AIN-treated L-ADSCs that was similar to that observed in response to treatment with short-term-cultured adipose-derived stem cells. CONCLUSIONS: This study suggests a novel method and strategy for cell-based tissue regeneration that can overcome the limitations of the low stemness and therapeutic efficacy of stem cells that occurs during long-term cell culture.

EGFR Fragmentation for Topological Transformation Nanobarcoding.

As the market for personalized lung cancer medicine expands, the demand for molecular diagnostic tools in general, and methods of detecting multiple genes with qualitative, quantitative, and high specificity in particular, have grown. Here, we propose a system for the effective detection of lung cancer-specific, long-length epidermal growth factor receptor (EGFR) gene mutations by using a topological transformation nano-barcoding technique (TNT). In former TNT studies, EGFR was successfully detected in cell environments and at test stages in the presence of a reference gene. However, because typical EGFR target concentrations are significantly lower at the clinical stage and the probe-binding ability of long-length targets is lower that of short targets, our system employs polymerase chain reaction (PCR) amplification, restriction, and filtering (PRF) for EGFR fragmentation to maximize performance. In a PRF system, the target is amplified by PCR, cut to a suitable size by a restriction enzyme, and filtered by a magnetic bead. With detection limits of 0.3555 % and 1.500 % for EGFR Del 19 and L858R mutations, respectively, the proposed TNT with PRF can effectively distinguish mutant cell lines and efficiently detect various lengths of genetic variations in clinical trials.

Improved Sensitivity of Intramolecular Strand Displacement Based on Localization of Probes.

Effective intermolecular interaction is required between probe and target molecules for successful detection of biomarkers. Here, we demonstrate that localization of probes on DNA nanostructures improves detection sensitivity and reaction rate. The structural flexibility of DNA nanostructures enabled frequent intramolecular interactions among the localized probes. The Smoluchowski coagulation method and the coarse-grained molecular dynamic software oxDNA were used for theoretical estimation of inter- and intramolecular behaviors of the DNA nanostructures as well as adequate experiments verifying the improvements in sensitivity with probe localization. Remarkably, the probe-localized DNA nanostructure had an increased sensitivity up to 274 times higher than that of the same probes without localization. We believe this achievement represents a wide applicability as a potential design strategy for robust, reliable, and sensitive biosensors.

Ultra-high curvature sensors for multi-bend structures using fiber Bragg gratings.

Measuring high curvatures is essential in various applications such as structural engineering, medical treatment, and robotics. Herein, we present a novel ultra-high curvature sensor with an ultra-thin and highly flexible structure incorporating fiber Bragg gratings (FBGs). The sensor can measure curvature of bidirectional bending up to 200 m-1. In addition, the multi-bend curvature sensor in conjunction with the proposed calibration scheme enables the accurate reconstruction of a curve with varying curvature. The sensitivity and the accuracy of the curvature sensor are investigated for different sensor designs. Finally, we demonstrate the accurate shape sensing of various 2-D patterns using the multi-bend curvatures sensor.

Protein Nanoparticle Fabrication for Optimized Reticuloendothelial System Evasion and Tumor Accumulation.

Nanoparticles (NPs) of protein-based materials have become one of the most promising candidates for drug carriers in drug-delivery systems because of their in vivo nontoxicity, biodegradability, compatibility with hydrophilic drugs, and adaptability to the human body. Many studies have investigated the fabrication of protein NPs from human serum albumin (HSA) as a new drug carrier. It is important for these NPs to remain in the blood until they reach their therapeutic target to achieve the desired effect; the quicker the clearance of drugs from the body, the shorter is the residence time of drugs in the body, which eventually reduces drug efficacy. Macrophage uptake is a major mechanism for clearance of NPs from the body, so, reducing the degree of macrophage uptake is a major challenge in drug-delivery systems. Original studies of HSA NP uptake by macrophages showed that denatured HSA and HSA NPs synthesized with 80% (v/v) ethanol showed a high degree of macrophage uptake. We found that HSA NPs synthesized with lower ethanol content at pH 7 showed lower macrophage uptake in in vitro macrophage cellular uptake experiments. The effects of the preparation parameters of ethanol concentration, pH, and glutaraldehyde on the macrophage uptake of NPs were thoroughly studied. This newly developed protein NP with lower macrophage uptake has potential application as a drug carrier for many delivery systems.

Revealing the Presence of a Symbolic Sequence Representing Multiple Nucleotides Based on K-Means Clustering of Oligonucleotides.

In biological systems, a few sequence differences diversify the hybridization profile of nucleotides and enable the quantitative control of cellular metabolism in a cooperative manner. In this respect, the information required for a better understanding may not be in each nucleotide sequence, but representative information contained among them. Existing methodologies for nucleotide sequence design have been optimized to track the function of the genetic molecule and predict interaction with others. However, there has been no attempt to extract new sequence information to represent their inheritance function. Here, we tried to conceptually reveal the presence of a representative sequence from groups of nucleotides. The combined application of the K-means clustering algorithm and the social network analysis theorem enabled the effective calculation of the representative sequence. First, a "common sequence" is made that has the highest hybridization property to analog sequences. Next, the sequence complementary to the common sequence is designated as a 'representative sequence'. Based on this, we obtained a representative sequence from multiple analog sequences that are 8⁻10-bases long. Their hybridization was empirically tested, which confirmed that the common sequence had the highest hybridization tendency, and the representative sequence better alignment with the analogs compared to a mere complementary.

Polymeric Nanocomplex Encapsulating Iron Oxide Nanoparticles in Constant Size for Controllable Magnetic Field Reactivity.

The magnetic properties of nanoparticles make them ideal for using in various applications, especially in biomedical applications. However, the magnetic force generated by a single nanoparticle is low. Herein, we describe the development of nanocomplexes (size of 100 nm) of many iron oxide nanoparticles (IONPs) encapsulated in poly(lactic- co-glycolic acid) (PLGA) using the simple method of emulsion solvent evaporation. The response of the IONP-encapsulated PLGA nanocomplexes (IPNs) to an external magnetic field could be controlled by modifying the amount of IONPs loaded into each nanocomplex. In a constant size of IPNs, larger loading numbers of IONPs resulted in more rapid responses to a magnetic field. In addition, nanocomplexes were coated with a silica layer to facilitate the addition of fluorescent dyes. This allowed visualization of the responses of the IPNs to an applied magnetic field corresponding to the IONP loading amount. We envision that these versatile, easy-to-fabricate IPNs with controllable magnetism will have important potential applications in diverse fields.

Cationic Surfactant-Induced Formation of Uniform Gold Nanoparticle Clusters with High Efficiency of Photothermal Conversion under Near-Infrared Irradiation.

A novel and simple method for the fabrication of gold nanoparticle (AuNP) clusters was introduced for use as an efficient near-infrared (NIR) photothermal agent. Cationic surfactants were employed to assemble AuNPs into clusters, during which polyvinylpyrrolidone (PVP) was used to stabilize the AuNP clusters. Through this manner, AuNP clusters with a uniform shape and a narrow size distribution (55.4 ± 5.0 nm by electron microscope) were successfully obtained. A mechanism for the formation of AuNP clusters was studied and proposed. Electrostatic interactions between AuNPs and cationic surfactants, hydrophobic interactions between hydrocarbon chains of cationic surfactants, and repulsive steric interactions of PVP were found to play an important role with regard to the formation mechanism. Photothermal effect in the NIR range of the AuNP clusters was demonstrated; results presented a highly efficient photothermal conversion (with a maximum η of 65%) of the AuNP clusters. The clusters could be easily coated by a silica layer, enabling their biocompatibility and colloidal stability in physiological fluids. The easy-to-fabricate AuNP clusters showed high potential of use as an NIR photothermal agent for cancer therapy.

Differences in DNA Probe-Mediated Aggregation Behavior of Gold Nanomaterials Based on Their Geometric Appearance.

Nanoparticles are used extensively to detect nucleic acid biomarkers due to their analytical applicability and sensitivity. Systems employing the surface plasmon resonance of gold nanomaterials are overwhelmingly considered to be candidates. The aggregation of gold nanomaterials mediated by the hybridization of target DNA at the interface causes a change in the surface plasmon resonance inherent in gold nanomaterials. Such changes can be measured by spectroscopy or even visualized by the naked eye, enabling effective and positive detection. The optical properties of gold nanoparticles are affected by their shape. The geometric appearance of the nanoparticles also affects their colloidal stability and aggregation behavior. In this study, we examined the effect of the geometric appearance of gold nanomaterials on DNA-mediated aggregation behavior through comparative experiments. Experimental and theoretical methods were used concurrently to derive accurate results and to support the hypotheses. Coarse-grained molecular dynamics simulations were performed with a large-scale atomic/molecular massively parallel simulator to understand the aggregation of nanoparticles with the same surface area and various aspect ratios. As a result, we confirmed that the aggregation sensitivity of nanoparticles was affected by the shape of the contact point with the gold nanomaterials. This study demonstrates that the design of a detection system should be accompanied by an in-depth review of the morphology of the nanoparticle.

Inhibitory effects of mesenchymal stem cells in intimal hyperplasia after balloon angioplasty.

OBJECTIVE: Intimal hyperplasia is a major cause of restenosis after arterial bypass and balloon angioplasty. Induction of rapid re-endothelialization has been proposed to reduce intimal hyperplasia. The aim of this study was to evaluate the inhibitory effect of mesenchymal stem cells on intimal hyperplasia. METHODS: Male New Zealand white rabbits were fed 1% cholesterol diets from 1 week before balloon angioplasty to the day of harvest. After dissection of rabbit carotid arteries, balloon angioplasty was performed with a 2F Fogarty embolectomy catheter. The injured carotid artery was coated with a mixture of 7 × 10(6) human umbilical cord mesenchymal stem cells (HUC-MSCs) and fibrin matrix. The carotid arteries were harvested 2, 4, and 8 weeks thereafter, and immunofluorescent staining and quantitative real-time polymerase chain reaction analysis were performed. RESULTS: The intima/media ratio was significantly reduced in the group treated with HUC-MSCs compared with the nontreated group (Student t-tests, *P < .05). The area of re-endothelialization was significantly higher (Student t-tests, *P < .05) in the group treated with HUC-MSCs than in the nontreated group. Expression of angiogenic genes such as vascular endothelial growth factor, platelet-derived growth factor, kinase insert domain receptor 1, angiopoietin 1, and angio-associated migratory cell protein was increased (analysis of variance, P < .05) in the group treated with HUC-MSCs relative to the nontreated group. CONCLUSIONS: Our study showed that HUC-MSCs reduce the formation of intimal hyperplasia through rapid re-endothelialization. This result might be applied to development of stem cell-coated stents as well as to development of a stem cell-containing sheet coat for inhibition of intimal hyperplasia after angioplasty or surgery.

Multiplexed labeling system for high-throughput cell sorting.

Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.

X-shaped DNA potentiates therapeutic efficacy in colitis-associated colon cancer through dual activation of TLR9 and inflammasomes.

BACKGROUND: Immunotherapy has been extensively pursed as a promising strategy for the treatment of cancer. Pattern-recognition receptors (PRRs) play important roles in triggering activation of innate and adaptive immunity. Therefore, agents that stimulate PRRs could be useful for cancer immunotherapy. We developed two kinds of X-shaped double-stranded oligodeoxynucleotides (X-DNA), a single unit of X-DNA (XS-DNA) composed of four strands of DNA and a ligated X-DNA complex (XL-DNA) formed by crosslinking each XS-DNA to the other, and investigated if they had immunostimulatory activity and could be applied to anti-cancer immunotherapy. METHODS: Activation of MAPKs and NF-κB was determined by immunoblotting in bone marrow-derived primary dendritic cells (BMDCs). Immune cytokines and co-stimulatory molecules were measured by ELISA and flow cytometry analysis. Anti-cancer efficacy was examined in an azoxymethane/dextran sulfate sodium-induced colitis-associated colon cancer mouse model. Association of X-DNA and TLR9 was determined by co-immunoprecipitation followed by immunoblotting. The involvement of TLR9 and inflammasomes was determined using TLR9- or caspase-1-deficient BMDCs. Inflammasome activation was examined by degradation of pro-caspase-1 to caspase-1 and cleavage of pro-IL-1ß to IL-1ß in BMDCs. RESULTS: XL-DNA and XS-DNA induced activation of MAPKs and NF-κB and production of immune cytokines and co-stimulatory molecules in BMDCs. BMDCs stimulated by XL-DNA induced differentiation of naïve CD4(+) T cells to TH1 cells. Intravenous injection of XL-DNA into mice resulted in increased serum IFN-γ and IL-12 levels, showing in vivo efficacy of XL-DNA to activate TH1 cells and dendritic cells. XL-DNA greatly enhanced the therapeutic efficacy of doxorubicin, an anti-cancer drug, in colitis-associated colon cancer. XL-DNA directly associated with TLR9. In addition, immunostimulatory activities of X-DNA were abolished in TLR9-deficient dendritic cells. Furthermore, X-DNA induced caspase-1 degradation and IL-1ß secretion in BMDCs, which were abolished in caspase-1-deficient cells. CONCLUSIONS: X-DNA induced the activation of dendritic cells as shown by the expression of immune-cytokines and co-stimulatory molecules, resulting in the differentiation of TH1 cells, mediated through dual activation of TLR9 and inflammasomes. X-DNA represents a promising immune adjuvant that can enhance the therapeutic efficacy of anti-cancer drugs by activating PRRs.

Evaluation of the formation of a junctional DNA nanostructure through annealing curve analysis.

During the self-assembly of different numbers of oligonucleotides comprising junctional DNA nanostructures, a change in environmental variables (e.g., temperature or salt concentration) has a substantial influence on the final products. Further, distinctive annealing temperatures of oligonucleotides are observed depending on the state of hybridization. Here, we present an evaluation of the annealing characteristics of oligonucleotides for the formation of a simple junctional DNA nanostructure using an annealing curve analysis. This method may be useful for analyzing the formation of complex junctional DNA nanostructures.

mRNA-Producing Pseudo-nucleus System.

A pseudo-eukaryotic nucleus (PEN) system consisting of a gene-containing DNA hydrogel encapsulated in a liposome is fabricated. Owing to the structural characteristics of gene-containing DNA hydrogel, mRNA transcription efficiency is promoted 2.57-fold. Through the use of PEN as a platform for mRNA delivery to the cytosol, prolonged protein translation is achieved.

X-DNA origami-networked core-supported lipid stratum.

DNA hydrogels are promising materials for various fields of research, such as in vitro protein production, drug carrier systems, and cell transplantation. For effective application and further utilization of DNA hydrogels, highly effective methods of nano- and microscale DNA hydrogel fabrication are needed. In this respect, the fundamental advantages of a core-shell structure can provide a simple remedy. An isolated reaction chamber and massive production platform can be provided by a core-shell structure, and lipids are one of the best shell precursor candidates because of their intrinsic biocompatibility and potential for easy modification. Here, we demonstrate a novel core-shell nanostructure made of gene-knitted X-shaped DNA (X-DNA) origami-networked gel core-supported lipid strata. It was simply organized by cross-linking DNA molecules via T4 enzymatic ligation and enclosing them in lipid strata. As a condensed core structure, the DNA gel shows Brownian behavior in a confined area. It has been speculated that they could, in the future, be utilized for in vitro protein synthesis, gene-integration transporters, and even new molecular bottom-up biological machineries.

Water-gel for gating graphene transistors.

Water, the primary electrolyte in biology, attracts significant interest as an electrolyte-type dielectric material for transistors compatible with biological systems. Unfortunately, the fluidic nature and low ionic conductivity of water prevents its practical usage in such applications. Here, we describe the development of a solid state, megahertz-operating, water-based gate dielectric system for operating graphene transistors. The new electrolyte systems were prepared by dissolving metal-substituted DNA polyelectrolytes into water. The addition of these biocompatible polyelectrolytes induced hydrogelation to provide solid-state integrity to the system. They also enhanced the ionic conductivities of the electrolytes, which in turn led to the quick formation of an electric double layer at the graphene/electrolyte interface that is beneficial for modulating currents in graphene transistors at high frequencies. At the optimized conditions, the Na-DNA water-gel-gated flexible transistors and inverters were operated at frequencies above 1 MHz and 100 kHz, respectively.

Observation of Ultrahigh Photoconductivity in DNA-MoS2 Nano-Biocomposite.

A nano-biocomposite film with ultrahigh photoconductivity remains elusive and critical for bio-optoelectronic applications. A uniform, well-connected, high-concentration nanomaterial network in the biological matrix remains challenging to achieve high photoconductivity. Wafer-scale continuous nano-biocomposite film without surface deformations and cracks plays another major obstacle. Here ultrahigh photoconductivity is observed in deoxyribonucleic acid-molybdenum disulfide (DNA-MoS2) nano-biocomposite film by incorporating a high-concentration, well-percolated, and uniform MoS2 network in the ss-DNA matrix. This is achieved by utilizing DNA-MoS2 hydrogel formation, which results in crack-free, wafer-scale DNA-MoS2 nano-biocomposite films. Ultra-high photocurrent (5.5 mA at 1 V) with a record-high on/off ratio (1.3 × 106) is observed, five orders of magnitude higher than conventional biomaterials (≈101) reported so far. The incorporation of the Wely semimetal (Bismuth) as an electrical contact exhibits ultrahigh photoresponsivity (2.6 × 105 A W-1). Such high photoconductivity in DNA-MoS2 nano-biocomposite could bridge the gap between biology, electronics, and optics for innovative biomedicine, bioengineering, and neuroscience applications.

A biospecies-derived genomic DNA hybrid gel electrolyte for electrochemical energy storage.

Intrinsic impediments, namely weak mechanical strength, low ionic conductivity, low electrochemical performance, and stability have largely inhibited beyond practical applications of hydrogels in electronic devices and remains as a significant challenge in the scientific world. Here, we report a biospecies-derived genomic DNA hybrid gel electrolyte with many synergistic effects, including robust mechanical properties (mechanical strength and elongation of 6.98 MPa and 997.42%, respectively) and ion migration channels, which consequently demonstrated high ionic conductivity (73.27 mS/cm) and superior electrochemical stability (1.64 V). Notably, when applied to a supercapacitor the hybrid gel-based devices exhibit a specific capacitance of 425 F/g. Furthermore, it maintained rapid charging/discharging with a capacitance retention rate of 93.8% after ∼200,000 cycles while exhibiting a maximum energy density of 35.07 Wh/kg and a maximum power density of 193.9 kW/kg. This represents the best value among the current supercapacitors and can be immediately applied to minicars, solar cells, and LED lightning. The widespread use of DNA gel electrolytes will revolutionize human efforts to industrialize high-performance green energy.