4. Publications
Full list of publications
https://scholar.google.com/citations?user=rpvienIAAAAJ&hl=en
Recent publications
Split-tracrRNA as an efficient tracrRNA system with an improved potential of scalability
Biomaterials Science 2023, 11, 3241-3251
Due to the relatively long sequence, tracrRNAs are chemically less synthesizable than crRNAs, leading to limited scalability of RNA guides for CRISPR-Cas9 systems. To develop shortened versions of RNA guides with improved cost-effectiveness, we have developed a split-tracrRNA system by nicking the 67-mer tracrRNA (tracrRNA(67)). Cellular gene editing assays and in vitro DNA cleavage assays revealed that the position of the nick is critical for maintaining the activity of tracrRNA(67). TracrRNA(41 + 23), produced by nicking in stem loop 2, showed gene editing efficiency and specificity comparable to those of tracrRNA(67). Removal of the loop of stem loop 2 was further possible without compromising the efficiency and specificity when the stem duplex was stabilized via a high GC content. Binding assays and single-molecule experiments suggested that efficient split-tracrRNAs could be engineered as long as their binding affinity to Cas9 and their reaction kinetics are similar to those of tracrRNA(67).
Cholesterol-Mediated Seeding of Protein Corona on DNA Nanostructures for Targeted Delivery of Oligonucleotide Therapeutics to Treat Liver Fibrosis
ACS Nano 2022, 16, 7331-7343
The protein corona is a protein layer formed on the surface of nanoparticles administered in vivo and considerably affects the in vivo fate of nanoparticles. Although it is challenging to control protein adsorption on nanoparticles precisely, the protein corona may be harnessed to develop a targeted drug delivery system if the nanoparticles are decorated with a ligand with enhanced affinity to target tissue- and cell-homing proteins. Here, we prepared a DNA tetrahedron with trivalent cholesterol conjugation (Chol3-Td) that can induce enhanced interaction with lipoproteins in serum, which in situ generates the lipoprotein-associated protein corona on a DNA nanostructure favorable for cells abundantly expressing lipoprotein receptors in the liver, such as hepatocytes in healthy mice and myofibroblasts in fibrotic mice. Chol3-Td was further adopted for liver delivery of antisense oligonucleotide (ASO) targeting TGF-β1 mRNA to treat liver fibrosis in a mouse model. The potency of ASO@Chol3-Td was comparable to that of ASO conjugated with the clinically approved liver-targeting ligand, trivalent N-acetylgalactosamine (GalNAc3), demonstrating the potential of Chol3-Td as a targeted delivery system for oligonucleotide therapeutics. This study suggests that controlled seeding of the protein corona on nanomaterials can provide a way to steer nanoparticles into the target area.
Kissing loop-mediated fabrication of RNA nanoparticles and their potential as cellular and in vivo siRNA delivery platforms
Biomaterials Science 2021, 9, 8148-8152
We describe an efficient method to condense RNAs into tightly packed RNA nanoparticles (RNPs) for biomedical applications without hydrophobic or cationic agents. We embedded kissing loops and siRNA in the RNAs to constrain the size of RNPs to ca. 100 nm, making them suitable not only for cellular uptake but also for passive tumor accumulation. The resulting RNPs were efficiently internalized into cells and downregulated the target gene of siRNAs. When intravenously injected into tumor-bearing mice, RNPs could also accumulate in the tumor. The reported fabrication method could be readily adopted as a platform to prepare RNPs for in vitro and in vivo delivery of bioactive RNAs.
https://pubs.rsc.org/en/content/articlehtml/2021/bm/d1bm01440d
Chimeric crRNAs Retaining Activity of Cas12a with Potential to Improve Specificity
Bulletin of the Korean Chemical Society, 2021, 42 (1), 87-90
RNA residues in the guide region of crRNA for Cas12a can be partially replaced by DNA.
https://onlinelibrary.wiley.com/doi/full/10.1002/bkcs.12109
Kidney-Targeted Cytosolic Delivery of siRNA Using a Small-Sized Mirror DNA Tetrahedron for Enhanced Potency
ACS Central Science 2020, 6 (12), 2250-2258
A proper intracellular delivery method with target tissue specificity is critical to utilize the full potential of therapeutic molecules including siRNAs while minimizing their side effects. Herein, we prepare four small-sized DNA tetrahedrons (sTds) by self-assembly of different sugar backbone-modified oligonucleotides and screened them to develop a platform for kidney-targeted cytosolic delivery of siRNA. An in vivo biodistribution study revealed the kidney-specific accumulation of mirror DNA tetrahedron (L-sTd). Low opsonization of L-sTd in serum appeared to avoid liver clearance and keep its size small enough to be filtered through the glomerular basement membrane (GBM). After GBM filtration, L-sTd could be delivered into tubular cells by endocytosis. The kidney preference and the tubular cell uptake property of the mirror DNA nanostructure could be successfully harnessed for kidney-targeted intracellular delivery of p53 siRNA to treat acute kidney injury (AKI) in mice. Therefore, L-sTd could be a promising platform for kidney-targeted cytosolic delivery of siRNA to treat renal diseases.
Systemic delivery of aptamer–drug conjugates for cancer therapy using enzymatically generated self-assembled DNA nanoparticles
Nanoscale 2020, 12 (45) 22945-22951
Aptamer–drug conjugates (ApDCs) are promising anticancer therapeutics with cancer cell specificity. However, versatile in vivo applications of ApDCs are hampered by their limited serum stability and inability to reach the tumour upon systemic administration. Here, we describe DNA nanoparticles of ApDCs as a platform for tumour-targeted systemic delivery of ApDCs. DNA nanoparticles of approximately 75 nm size were fabricated by self-assembly of a polymerised floxuridine (FUdR)-incorporated AS1411 aptamer produced via rolling circle amplification. The DNA nanoparticles of ApDCs showed highly efficient cancer cell uptake, enhanced serum stability, and tumour-targeted accumulation. These properties could be successfully utilised for tumour-specific apoptotic damage by ApDCs, leading to significant suppression of tumour growth without considerable systemic toxicity. Molecular analysis revealed that the enhanced anticancer potency was due to the synergic effect induced by the simultaneous activation of p53 by AS1411 and the inhibition of thymidylate synthase by FUdR, respectively, both of which were generated from the DNA nanoparticles. We therefore expect that the DNA nanoparticles of ApDCs can be a promising platform for tumour-targeted delivery of various nucleoside-incorporated ApDCs to treat cancer.
Lung-targeted delivery of TGF-β antisense oligonucleotides to treat pulmonary fibrosis
Journal of Controlled Release 2020, 32, 108-121
Pulmonary fibrosis is a serious respiratory disease, with limited therapeutic options. Since TGF-β is a critical factor in the fibrotic process, downregulation of this cytokine has been considered a potential approach for disease treatment. Herein, we designed a new lung-targeted delivery technology based on the complexation of polymeric antisense oligonucleotides (pASO) and dimeric human β-defensin 23 (DhBD23). Antisense oligonucleotides targeting TGF-β mRNA were polymerized by rolling circle amplification and complexed with DhBD23. After complexation with DhBD23, pASO showed improved serum stability and enhanced uptake by fibroblasts in vitro and lung-specific accumulation upon intravenous injection in vivo. The pASO/DhBD23 complex delivered into the lung downregulated target mRNA, and subsequently alleviated lung fibrosis in mice, as demonstrated by western blotting, quantitative reverse-transcriptase PCR (qRT-PCR), immunohistochemistry, and immunofluorescence imaging. Moreover, as the complex was prepared only with highly biocompatible materials such as DNA and human-derived peptides, no systemic toxicity was observed in major organs. Therefore, the pASO/DhBD23 complex is a promising gene therapy platform with lung-targeting ability to treat various pulmonary diseases, including pulmonary fibrosis, with low side effects.
The crystal structure of a natural DNA polymerase complexed with mirror DNA
Chemical Communications 2020, 56, 2186-2189
The intrinsic L-DNA binding properties of a natural DNA polymerase was discovered. The binding affinity of Dpo4 polymerase for L-DNA was comparable to that for D-DNA. The crystal structure of Dpo4/L-DNA complex revealed a dimer formed by the little finger domain that provides a binding site for L-DNA.
A self-assembled DNA tetrahedron as a carrier for in vivo liver-specific delivery of siRNA
Biomaterials Science 2020, 8, 586-590
While siRNA is a potent therapeutic tool that can silence disease-causing mRNA, its in vivo potency can be compromised due to lack of target tissue specificity. Here, we report a wireframe tetrahedral DNA nanostructure having a 20-mer duplex on each side that can be specifically distributed into the liver upon systemic administration. This liver-targeted DNA tetrahedron is employed as the carrier for liver-specific delivery of siRNA targeting ApoB1 mRNA, which is overexpressed in hypercholesterolemia. When delivered by DNA tetrahedron, the siRNA can preferentially be accumulated in liver and down-regulate the ApoB1 protein. As a result, the blood cholesterol level is also decreased by the siRNA. These results successfully demonstrate that the DNA tetrahedron is a promising carrier for liver-targeted delivery of therapeutic nucleic acids.
L-DNA linear duplex: An efficient drug delivery carrier with a simple structure
Journal of Industrial and Engineering Chemistry 2019, 74, 187-192
Three dimensional DNA nanostructures are often limited in scalability due to low assembly yield at high concentrations, leaving themselves less suitable for industrial uses. To overcome this drawback, we here employed L-DNA duplex (L-ds) as a drug carrier. Due to its simple structure, L-ds can easily be prepared at micromolar concentrations without causing any considerable by-product, providing sufficient scalability for practical development. With sufficient cellular uptake efficiency in SCC7 cells and the tumor accumulation level in a SCC7-tumor xenograft mouse model, L-ds could well be utilized as a carrier for intracellular and tumor delivery of an anticancer agent, providing substantial cytotoxicity and inhibition of tumor growth, respectively.
Chimeric crRNAs with 19 DNA residues in the guide region show the retained DNA cleavage activity of Cas9 with potential to improve the specificity
Chemical Communications 2019, 55, 3552-3555
We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents. The cellular activity of the chimeric crRNAs to disrupt the target gene was comparable to that of the native crRNA.
Highly tumor-specific DNA nanostructures discovered by in vivo screening of a nucleic acid cage library and their applications in tumor-targeted drug delivery
Biomaterials 2019, 195, 1-12
Enormous efforts have been made to harness nanoparticles showing extravasation around tumors for tumor-targeted drug carriers. Owing to the complexity of in vivo environments, however, it is very difficult to rationally design a nanoconstruct showing high tumor specificity. Here, we show an approach to develop tumor-specific drug carriers by screening a library of self-assembled nucleic acid cages in vivo. After preparation of a library of 16 nucleic acid cages by combining the sugar backbone and the shape of cages, we screened the biodistribution of the cages intravenously injected into tumor-bearing mice, to discover the cages with high tumor-specificity. This tumor specificity was found to be closely related with serum stability, cancer cell uptake efficiency, and macrophage evasion rate. We further utilized the cages showing high tumor specificity as carriers for the delivery of not only a cytotoxic small molecule drug but also a macromolecular apoptotic protein exclusively into the tumor tissue to induce tumor-specific damage. The results demonstrate that our library-based strategy to discover tumor-targeted carriers can be an efficient way to develop anti-cancer nanomedicines with tumor specificity and enhanced potency.
Modulating α-synuclein fibril formation using DNA tetrahedron nanostructures
Biophys Acta Gen Subj. 2019, 1863(1):73-81
The small presynaptic protein α-synuclein (α-syn) is involved in the etiology of Parkinson's disease owing to its abnormal misfolding. To date, little information is known on the role of DNA nanostructures in the formation of α-syn amyloid fibrils. Here, the effects of DNA tetrahedrons on the formation of α-syn amyloid fibrils were investigated using various biochemical and biophysical methods such as thioflavin T fluorescence assay, atomic force microscopy, light scattering, transmission electron microscopy, and cell-based cytotoxicity assay. It has been shown that DNA tetrahedrons decreased the level of oligomers and increased the level of amyloid fibrils, which corresponded to decreased cellular toxicity. The ability of DNA tetrahedron to facilitate the formation of α-syn amyloid fibrils demonstrated that structured nucleic acids such as DNA tetrahedrons could modulate the process of amyloid fibril formation. Our study suggests that DNA tetrahedrons could be used as an important facilitator toward amyloid fibril formation of α-synuclein, which may be of significance in finding therapeutic approaches to Parkinson's disease and related synucleinopathies.
Tetrahedral DNAzymes for enhanced intracellular gene-silencing activity
Chem. Commun. 2018, 54, 9410.
We prepared tetrahedral DNAzymes (TDzs) to overcome potential limitations such as insufficient serum stability and poor cellular uptake of single-stranded DNAzymes (ssDzs). TDzs showed enhanced serum stability and higher cellular uptake efficiency than those of ssDzs, providing significantly improved intracellular gene-silencing activity to down-regulate the target mRNA level.
Shaping Rolling Circle Amplification Products into DNA Nanoparticles by Incorporation of Modified Nucleotides and Their Application to In Vitro and In Vivo Delivery of a Photosensitizer
Molecules 2018, 23(7), 1833;
Rolling circle amplification (RCA) is a robust way to generate DNA constructs, which are promising materials for biomedical applications including drug delivery because of their high biocompatibility. To be employed as a drug delivery platform, however, the DNA materials produced by RCA need to be shaped into nanoparticles that display both high cellular uptake efficiency and nuclease resistance. Here, we showed that the DNA nanoparticles (DNPs) can be prepared with RCA and modified nucleotides that have side-chains appended on the nucleobase are capable of interacting with the DNA strands of the resulting RCA products. The incorporation of the modified nucleotides improved cellular uptake efficiency and nuclease resistance of the DNPs. We also demonstrated that these DNPs could be employed as carriers for the delivery of a photosensitizer into cancer cells to achieve photodynamic therapy upon irradiation at both the in vitro and in vivo levels.
Dimeric Human β-Defensin 3 as a Universal Platform for Intracellular Delivery of Nucleic Acid Cargos
ACS Appl. Bio Mater., 2018, 1, 100-109
Functional nucleic acids including siRNA, mRNA, and plasmid DNA are promising bioactive molecules to regulate cellular functions uncontrollable by conventional small molecule regulators. To realize successful cellular applications of these nucleic acids, an intracellular gene delivery vehicle with high efficiency and low cytotoxicity is required. Here, we report the dimerization of human β-defensin 3 (DhBD3) promoted by the interaction between β-strands and the application of DhBD3 for efficient delivery of various nucleic acid cargos. DhBD3 with multiple cationic residues could be complexed with various types of polyanionic DNA and RNA. DhBD3 could intracellularly deliver both small and large nucleic acid cargos loaded by complexation to regulate the expression level of target proteins, showing its potential as a universal platform for nucleic acid delivery. In addition, as DhBD3 is a human-derived material with high biocompatibility and can be robustly prepared by an inexpensive method, it is a promising gene delivery system that can be employed for biomedical purposes.
Streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular and tumor delivery of enzymes
Despite the extremely high substrate specificity and catalytically amplified activity of enzymes, the lack of efficient cellular internalization limits their application as therapeutics. To overcome this limitation and to harness enzymes as practical biologics for targeting intracellular functions, we developed the streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular delivery of various enzymes. The hybrid consists of streptavidin, which provides a stoichiometrically controlled loading site for the enzyme cargo and an L-DNA (mirror DNA) tetrahedron, which provides the intracellular delivery potential. Due to the cell-penetrating ability of the mirror DNA tetrahedron of this hybrid, enzymes loaded on streptavidin can be efficiently delivered into the cells, intracellularly expressing their activity. In addition, we demonstrate tumor delivery of enzymes in an animal model by utilizing the potential of the hybrid to accumulate in tumors. Strikingly, the hybrid is able to transfer the apoptotic enzyme specifically into tumor cells, leading to strong suppression of tumor growth without causing significant damage to other tissues. These results suggest that the hybrid may allow anti-proliferative enzymes and proteins to be utilized as anticancer drugs.
Reversible Regulation of Enzyme Activity by pH-Responsive Encapsulation in DNA Nanocages
Reversible regulation of enzyme activity by chemical and physical stimuli is often achieved by incorporating stimuli-responsive domains in the enzyme of interest. However, this method is suitable for a limited number of enzymes with well-defined structural and conformational changes. In this study, we present a method to encapsulate enzymes in a DNA cage that could transform its conformation depending on the pH, allowing reversible control of the accessibility of the enzyme to the surrounding environment. This enabled us to regulate various properties of the enzyme, such as its resistance to protease-dependent degradation, binding affinity to the corresponding antibody, and most importantly, enzyme activity. Considering that the size and pH responsiveness of the DNA cage can be easily adjusted by the DNA length and sequence, our method provides a broad-impact platform for controlling enzyme functions without modifying the enzyme of interest.
In vitro and in vivo behavior of DNA tetrahedrons as tumor-targeting nanocarriers for doxorubicin delivery
Deoxyribonucleic acid (DNA) is a versatile material with high applicability and inherent biocompatibility. L-DNA, the perfect mirror form of the naturally occurring D-DNA, has been used in DNA nanotechnology. It has thermodynamically identical properties to D-DNA, is capable of self-assembly and bio-orthogonal base-pairing, and is resistant to nuclease activity. We previously constructed an L-DNA tetrahedron (L-Td) and found that this nanostructure has remarkably higher capacity for cell penetration than its natural counterpart (D-Td). L-Td molecules of two different sizes-one with 17-mer per side (L-Td17) and the other with 30-mer per side (L-Td30)-were prepared by assembling four L-DNA strands. In this study, cellular uptake of L-Td with different sizes was observed over time using a laser scanning confocal microscope (LSCM) equipped with a live cell chamber system. In addition, we conducted a pharmacokinetic study to examine the potential of L-Td as a carrier for in vivo tumor-targeted delivery of a low dose of doxorubicin (DOX). L-Td entered into the cells through endocytosis, and a specific DNA sequence of the L-Td ensures targeted entry into cancer cells. Compared with free DOX, DOX-loaded L-Td (DOX@L-Td) showed decreased clearance and increased initial concentration (C0), half-life, and area under the curve (AUC), indicating that DOX@L-Td circulated in the blood stream for longer than free DOX. L-Td17, in particular, had beneficial effects owing to its ability to enhance tumor accumulation of DOX and reduce the cardiotoxicity caused by it through administration of a low dose of the drug.
Backbone-modified oligonucleotides for tuning the cellular uptake behaviour of spherical nucleic acids.
Biomater. Sci. 2017 Feb 28;5(3):412-416.
Spherical nucleic acids (SNAs) are spherically arranged oligonucleotides on core inorganic nanoparticles and have great potential for intracellular delivery of bioactive molecules, since they have been found to be internalized into mammalian cells. Understanding the factors that influence the cellular uptake of SNAs would be beneficial to design SNAs with novel uptake properties. We here report the effect of the sugar backbone type of the oligonucleotides on the cellular internalization of SNAs. After the preparation of SNAs with the oligonucleotides of five different sugar backbones, we analyze the cellular uptake efficiency quantitatively by flow cytometry and inductively coupled plasma mass spectrometry (ICP-MS). The data reveal that the uptake efficiencies and the uptake mechanisms significantly rely on the backbone type. These results suggest that the backbone modification can provide a unique handle to tune the cellular uptake behavior of SNAs.
Poly-sgRNA/siRNA ribonucleoprotein nanoparticles for targeted gene disruption.
J. Control. Release 2017, 250, 27-35
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) can be used for the specific disruption of a target gene to permanently suppress the expression of the protein encoded by the target gene. Efficient delivery of the system to an intracellular target site should be achieved to utilize the tremendous potential of the genome-editing tool in biomedical applications such as the knock-out of disease-related genes and the correction of defect genes. Here, we devise polymeric CRISPR/Cas9 system based on poly-ribonucleoprotein (RNP) nanoparticles consisting of polymeric sgRNA, siRNA, and Cas9 endonuclease in order to improve the delivery efficiency. When delivered by cationic lipids, the RNP nanoparticles built with chimeric poly-sgRNA/siRNA sequences generate multiple sgRNA-Cas9 RNP complexes upon the Dicer-mediated digestion of the siRNA parts, leading to more efficient disruption of the target gene in cells and animal models, compared with the monomeric sgRNA-Cas9 RNP complex.
http://www.sciencedirect.com/science/article/pii/S0168365916309075
Modular delivery of CpG-incorporated lipid-DNA nanoparticles for spleen DC activation.
Biomaterials. 2017 Jan;115:81-89.
We introduce a versatile carrier system for in vitro and in vivo immune stimulation based on soft matter DNA nanoparticles (NPs). The incorporation of lipid-modified nucleotides into DNA strands enables the formation of micelles of uniform size. In a single self-assembly step, the micelles can be equipped with immune adjuvant (CpG) motifs and fluorescent probes. The immunological effects of CpG confined at the NP surface were studied in a comprehensive manner in animal experiments. Dose-dependent activation of spleen dendritic cells (DCs) by CpG-conjugated NP was observed, which was accompanied by the pronounced up-regulation of co-stimulatory molecule and cytokine production.
Self-assembled mirror DNA nanostructures for tumor-specific delivery of anticancer drugs
J. Control. Release 2016, 243:121-131.
Nanoparticle delivery systems have been extensively investigated for targeted delivery of anticancer drugs over the past decades. However, it is still a great challenge to overcome the drawbacks of conventional nanoparticle systems such as liposomes and micelles. Various novel nanomaterials consist of natural polymers are proposed to enhance the therapeutic efficacy of anticancer drugs. Among them, deoxyribonucleic acid (DNA) has received much attention as an emerging material for preparation of self-assembled nanostructures with precise control of size and shape for tailored uses. In this study, self-assembled mirror DNA tetrahedron nanostructures is developed for tumor-specific delivery of anticancer drugs. l-DNA, a mirror form of natural d-DNA, is utilized for resolving a poor serum stability of natural d-DNA. The mirror DNA nanostructures show identical thermodynamic properties to that of natural d-DNA, while possessing far enhanced serum stability. This unique characteristic results in a significant effect on the pharmacokinetics and biodistribution of DNA nanostructures. It is demonstrated that the mirror DNA nanostructures can deliver anticancer drugs selectively to tumors with enhanced cellular and tissue penetration. Furthermore, the mirror DNA nanostructures show greater anticancer effects as compared to that of conventional PEGylated liposomes. Our new approach provides an alternative strategy for tumor-specific delivery of anticancer drugs and highlights the promising potential of the mirror DNA nanostructures as a novel drug delivery platform.
Performance of a novel fluorogenic chimeric analog for the detection of third-generation cephalosporin resistant bacteria.
Resistance to third generation cephalosporins is widely disseminated in Enterobacteriaceae mainly due to extended-spectrum-β-lactamases, plasmid AmpC β-lactamases, and hyperproduction of chromosomal AmpC β-lactamases. Here we evaluated the performance of a novel fluorogenic probe rapid test and compared the results with the phenol red assay using a total of 77 characterized organisms (44 extended-spectrum-β-lactamases, 33 chromosomal or plasmid AmpC β-lactamases) and 46 susceptible organisms. The fluorescent assay showed higher sensitivity than the phenol red assay in cefotaximase type extended-spectrum-β-lactamases, non- cefotaximase type extended-spectrum-β-lactamases, chromosomal AmpC β-lactamases, and plasmid AmpC β-lactamases (96.7% vs. 90.0%, p=0.157; 71.4% vs. 7.1%, p=0.003; 100.0% vs. 64.7%, p<0.001; 100.0% vs. 6.3%, p<0.001). The fluorescent assay had a positive correlation with the exponents of cefotaxime and ceftazidime minimum inhibitory concentrations (p<0.001 for both). The new fluorescent assay will be very useful for the rapid detection of resistance to third generation cephalosporins that originates from various β-lactamases.
http://www.sciencedirect.com/science/article/pii/S0167701216303049
Biophysical and chemical handles to control the size of DNA nanoparticles produced by rolling circle amplification
Although rolling circle amplification (RCA) is an efficient method to produce DNA materials for biomedical applications, it does not yield nano-sized products suitable for intracellular delivery. We here provide the ways to control the size of RCA products and show a potential application of the size-controlled DNA nanoparticles.
A fluorogenic substrate of beta-lactamases and its potential as a probe to detect the bacteria resistant to the third-generation oxyimino-cephalosporins
We devised and synthesized a fluorogenic substrate of β-lactamases as a probe to detect the activity of the enzymes. Fluorescence of the probe emitted upon treatment of a β-lactamase and increased proportionally to the concentration of the enzyme, demonstrating its sensing property for the activity of the enzyme. We also showed that the probe could be utilized to assay the enzyme and to determine kinetic parameters of the enzyme. Moreover, the probe was able to detect resistance to the third-generation oxyimino-cephalosporin-derived antibiotics such as cefotaxime and ceftazidime. In particular, the probe could identify the ceftazidime-resistance in bacteria that was not detectable using conventional pH-sensing materials, indicating the practical utility of the probe.
http://www.sciencedirect.com/science/article/pii/S095656631530542X
Utilization of a pH‐Responsive DNA Motif for β‐Lactamase Assays
Bulletin of the Korean Chemical Society 2016 Oct. 37 (10), 1582-1585
http://onlinelibrary.wiley.com/doi/10.1002/bkcs.10698/full
Overcoming doxorubicin resistance of cancer cells by Cas9-mediated gene disruption
In this study, Cas9 system was employed to down-regulate mdr1 gene for overcoming multidrug resistance of cancer cells. Disruption of the MDR1 gene was achieved by delivery of the Cas9-sgRNA plasmid or the Cas9-sgRNA ribonucleoprotein complex using a conventional gene transfection agent and protein transduction domain (PTD). Doxorubicin showed considerable cytotoxicity to the drug-resistant breast cancer cells pre-treated with the RNA-guided endonuclease (RGEN) systems, whereas virtually non-toxic to the untreated cells. The potency of drug was enhanced in the cells treated with the protein-RNA complex as well as in those treated with plasmids, suggesting that mutation of the mdr1 gene by intracellular delivery of Cas9-sgRNA complex using proper protein delivery platforms could recover the drug susceptibility. Therefore, Cas9-mediated disruption of the drug resistance-related gene can be considered as a promising way to overcome multidrug resistance in cancer cells.
http://www.nature.com/articles/srep22847
Nano-formulation of a photosensitizer using a DNA tetrahedron and its potential for in vivo photodynamic therapy
Photodynamic therapy (PDT) is a cytotoxic treatment using singlet oxygen produced by photosensitizers. Approved porphyrinoid PDT still suffers from a lack of robust production methods and low water solubility. Methylene blue (MB) is a good candidate for the PDT drug, because the dye is an effective photosensitizer, can be easily synthesized, and is already being used in other clinical fields. However, its poor cell/tissue penetration and low stability against the reducible biological conditions should be addressed by using a proper delivery vehicle. Here, we employed a DNA tetrahedron, a self-assembled nanostructure as the carrier for intracellular delivery of MB by taking advantage of the DNA binding property of the photosensitizer and demonstrated photo-induced cytotoxicity by the MB delivered by the DNA nanocarrier. We also evaluated the PDT potency of the MB-loaded DNA nanoconstruct in vivo tumor model to suppress tumor growth.
http://pubs.rsc.org/en/Content/ArticleLanding/2016/BM/C5BM00467E#!divAbstract
Fluorogenic nanoreactor assembly with boosted sensing kinetics for timely imaging of cellular hydrogen peroxide
Chem Commun (Camb). 2016 Jan 21;52(6):1131-4. doi: 10.1039/c5cc06387f
The precise detection of endogenous H2O2 has been considered to be a useful tool for understanding cell physiology. Here, we have developed a nanoreactor co-incorporated with a H2O2-responsive fluorogenic molecule and a catalytic additive. The fast sensing kinetics allows us to visualize a subcellular response in real-time.
http://pubs.rsc.org/en/Content/ArticleLanding/2016/CC/C5CC06387F#!divAbstract
A dual-responsive pH-sensor and its potential as a universal probe for assays of pH-changing enzymes
Analyst. 2015 Apr 21;140(8):2804-9. doi: 10.1039/c4an01844c
We described a dual turn-on probe sensitive to both acidity and basicity, which could be designed by connecting a fluorophore to a quencher via metal-ligand interaction. Atto488-labeled nitrilotriacetic acid and polyhistidine peptide were used as the fluorophore and the quencher, respectively, and linked to each other by coordination with a cobalt(II) ion. After preparation of the probe, the pH-sensitive dual turn-on property of the probe has been successfully observed upon responding to both acidity and basicity of the solution. The probe has been employed as a signal reporter in assays of pH-changing enzymes such as penicillinase generating acidity and adenosine deaminase generating basicity. Furthermore, the practical utility of the probe was also demonstrated by utilizing the probe in the discrimination of β-lactamase-producing bacteria.
http://pubs.rsc.org/en/Content/ArticleLanding/2015/AN/C4AN01844C#!divAbstract
A microwell plate-based multiplex immunoassay for simultaneous quantitation of antibodies to infectious viruses
Analyst. 2015 Mar 21;140(6):1995-2000. doi: 10.1039/c4an02262a
Antibodies (Abs) to disease-causing viruses in human blood are important indicators of infection status. While ELISA has been widely used to detect these Abs, a multiplex assay system for simultaneous detection of multiple Abs is still a desirable alternative method for a more efficient screening process because of the lack of multiplexing ability in ELISA. However, as all antibodies are based on immunoglobulin and recognized commonly by the same secondary antibody, it is impossible to multiplex the conventional indirect ELISA in a 96-microwell plate-based platform. To overcome this hurdle, we designed an assay consisting of two steps: capturing target Abs by specific antigens on DNA-encoded gold nanoparticles; and quantifying the target Abs by producing RNase H-mediated detection signals based on the DNA and additional RNA probes. With this newly designed method, we could simultaneously analyze three infectious disease-related Abs, anti-HIV Ab, anti-HCV Ab, and anti-HBV Ab, on the microwell-based platform. The assay performance was evaluated by comparison with ELISA. Furthermore, the accuracy and precision of the assay in a practical application was also estimated by determining the amount of target Abs in human serum solutions.
http://pubs.rsc.org/en/Content/ArticleLanding/2015/AN/C4AN02262A#!divAbstract
Discovery of a non-cationic cell penetrating peptide derived from membrane-interacting human proteins and its potential as a protein delivery carrier
Sci Rep. 2015 Jun 26;5:11719. doi: 10.1038/srep11719
Cell penetrating peptides (CPPs) are peptides that can be translocated into cells and used as a carrier platform for the intracellular uptake of cargo molecules. Subject to the source of CPP sequences and their positively charged nature, the cytotoxicity and immunogenicity of conventional CPPs needs to be optimized to expand their utility for biomedical applications. In addition to these safety issues, the stability of CPPs needs to be addressed since their positively charged residues are prone to interact with the biological milieu. As an effort to overcome these limitations of the current CPP technology, we isolated CPP candidate sequences and synthesized peptides from twelve isoforms of annexin, a family of membrane-interacting human proteins. The candidate screen returned a CPP rich in hydrophobic residues that showed more efficient cellular uptake than TAT-CPP. We then investigated the uptake mechanism, subcellular localization, and biophysical properties of the newly found CPP, verifying low cytotoxicity, long-term serum stability, and non-immunogenicity. Finally, model proteins conjugated to this peptide were successfully delivered into mammalian cells both in vitro and in vivo, indicating a potential use of the peptide as a carrier for the delivery of macromolecular cargos.
http://www.nature.com/articles/srep11719
Highly sensitive detection of a bio-threat pathogen by gold nanoparticle-based oligonucleotide-linked immunosorbent assay
Biosens Bioelectron. 2015 Feb 15;64:69-73. doi: 10.1016/j.bios.2014.08.038
Francisella (F.) tularensis causes the zoonotic disease tularemia and categorized as one of the highest-priority biological agents. The sensing approaches utilized by conventional detection methods, including enzyme-linked immunosorbent assay (ELISA), are not sensitive enough to identify an infectious dose of this high-risk pathogen due to its low infective dose. As an attempt to detect F. tularensis with high sensitivity, we utilized the highly sensitive immunoassay system named gold nanoparticle-based oligonucleotide-linked immunosorbent assay (GNP-OLISA) which uses antibody-gold nanoparticles conjugated with DNA strands as a signal generator and RNA oligonucleotides appended with a fluorophore as a quencher for signal amplification. We modified the GNP-OLISA for the detection F. tularensis to utilize one antibody for both the capture of the target and for signal generation instead of using two different antibodies, which are usually employed to construct the antibody sandwich in the ELISA. The GNP-OLISA showed 37-fold higher sensitivity compared with ELISA and generated very consistent detection results in the sera. In addition, the detection specificity was not affected by the presence of non-target bacteria, suggesting that GNP-OLISA can be used as a sensitive detection platform for monitoring high-risk pathogens thereby overcoming the limit of the conventional assay system.
http://www.sciencedirect.com/science/article/pii/S0956566314006265
Utilizing the bioorthogonal base-pairing system of L-DNA to design ideal DNA nanocarriers for enhanced delivery of nucleic acid cargos
Chem. Sci., 2014, 5, 1533-1537. DOI: 10.1039/C3SC52601A (Edge Article)
DNA nanoconstructs are a potential drug carrier with high biocompatibility. They are promising particularly when therapeutic nucleic acids are the cargos to be delivered, since both the carrier and the cargo are nucleic acids which can be designed, synthesized and assembled in a seamless feature without using post-synthetic conjugation chemistry. However, the unwanted base-paring events between the cargo and the carrier may potentially disturb the desired structure of the cargo-loaded carrier. To address this concern, we propose a DNA nanocarrier composed of L-DNA strands having a bioorthogonal base-paring system. The study presented here provides useful properties of L-DNA as a backbone for the DNA nanocarrier and demonstrates superiority of L-DNA over the natural D-DNA backbone in the delivery of an anti-proliferative aptamer as well as in the construction of the cargo-carrier assembly.
http://pubs.rsc.org/en/content/articlehtml/2014/sc/c3sc52601a#fn1
Correction: In the paper, the figure legend to Fig. 1d written as “(d) CD spectra of D-Td (blue) and L-Td (red)” should be corrected as follows: “(d) CD spectra of D-Td (red) and L-Td (blue)”.
Drug delivery by a self-assembled DNA tetrahedron for overcoming drug resistance in breast cancer cells
Chem Commun (Camb). 2013 Mar 11;49(20):2010-2. doi
A DNA tetrahedron is employed for efficient delivery of doxorubicin into drug-resistant breast cancer cells. The drug delivered with the DNA nanoconstruct is considerably cytotoxic, whereas free doxorubicin is virtually non-cytotoxic for the drug-resistant cells. Thus, the DNA tetrahedron, made of the inherently natural and biocompatible material, can be a good candidate for the drug carrier to overcome MDR in cancer cells.
http://pubs.rsc.org/en/Content/ArticleLanding/2013/CC/c3cc38693g#!divAbstract
Sentinel lymph node imaging by a fluorescently labeled DNA tetrahedron
Biomaterials. 2013 Jul;34(21):5226-35. doi: 10.1016/j.biomaterials.2013.03.074
Sentinel lymph nodes (SLNs) are the first lymph nodes which cancer cells reach after traveling through lymphatic vessels from the primary tumor. Evaluating the nodal status is crucial in accurate staging of human cancers and accordingly determines prognosis and the most appropriate treatment. The commonly used methods for SLN identification in clinics are based on employment of a colloid of radionuclide or injection of a small dye. Although these methods have certainly contributed to improve surgical practice, new imaging materials are still required to overcome drawbacks of the techniques such as inconvenience of handling radioactive materials and short retention time of small dyes in SLNs. Here, we prepare a fluorescence-labeled DNA tetrahedron and perform SLN imaging by using the DNA nanoconstruct. With a successful identification of SLNs by the DNA nanoconstruct, we suggest that DNA tetrahedron hold great promises for clinical applications.
