The positional and numerical effect of N 6-methyladenosine in tracrRNA on the DNA cleavage activity of Cas9.

Post-transcriptional modifications on the guide RNAs utilized in the Cas9 system may have the potential to impact the activity of Cas9. In this study, we synthesized a series of tracrRNAs containing N 6-methyadenosine (m6A), a prevalent post-transcriptional modification, at various positions. We evaluated the effect of these modifications on the DNA cleavage activity of Cas9. Our results show that multiple m6As in the anti-repeat region of tracrRNA reduce the DNA cleavage activity of Cas9. This suggests that the m6A-modified tracrRNA can be used for Cas9 only when the number and the position of the modified residue are properly chosen in tracrRNA.

Human ß-Defensin 23 as a Carrier for In Vitro and In Vivo Delivery of mRNA.

The successful application of mRNA therapeutics hinges on the effective intracellular delivery of mRNA both in vitro and in vivo. However, this remains a formidable challenge due to the polyanionic nature, longitudinal shape, and low nuclease resistance of mRNA. In this study, we introduce a novel mRNA delivery platform utilizing a human ß-defensin peptide, hBD23. The positive charge of hBD23 allows it to form nanocomplexes with mRNA, facilitating cellular uptake and providing protection against serum nucleases. When optimized for peptide-to-mRNA (N/P) ratios, these hBD23/mRNA complexes demonstrated efficient cellular delivery and subsequent protein expression both in vitro and in vivo. Importantly, as hBD23 is human derived, the complexes exhibited minimal cytotoxicity and immunogenicity. Given its high biocompatibility and delivery efficiency, hBD23 represents a promising platform for the in vitro and in vivo delivery of mRNA.

Cholesterol-Mediated Seeding of Protein Corona on DNA Nanostructures for Targeted Delivery of Oligonucleotide Therapeutics to Treat Liver Fibrosis.

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-ß1 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.

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.

Outcomes of critically ill patients according to the perception of intensivists on the appropriateness of intensive care unit admission.

BACKGROUND: It is important for intensivists to determine which patient may benefit from intensive care unit (ICU) admission. We aimed to assess the outcomes of patients perceived as non-beneficially or beneficially admitted to the ICU and evaluate whether their prognosis was consistent with the intensivists' perception. METHODS: A prospective observational study was conducted on patients admitted to the medical ICU of a tertiary referral center between February and April 2014. The perceptions of four intensivists at admission (day 1) and on day 3 were investigated as non-beneficial admission, beneficial admission, or indeterminate state. RESULTS: A total of 210 patients were enrolled. On days 1 and 3, 22 (10%) and 23 (11%) patients were judged as having non-beneficial admission; 166 (79%) and 159 (79%), beneficial admission; and 22 (10%) and 21 (10%), indeterminate state, respectively. The ICU mortality rates of each group on day 1 were 59%, 23%, and 59%, respectively; their 6-month mortality rates were 100%, 48%, and 82%, respectively. The perceptions of non-beneficial admission or indeterminate state were the significant predictors of ICU mortality (day 3: odds ratio [OR], 4.049; 95% confidence interval [CI], 1.892-8.664; P<0.001) and 6-month mortality (day 1: OR, 4.983; 95% CI, 1.260-19.703; P=0.022; day 3: OR, 4.459; 95% CI, 1.162-17.121; P=0.029). CONCLUSIONS: The outcomes of patients perceived as having non-beneficial admission were extremely poor. The intensivists' perception was important in predicting patients' outcomes and was more consistent with long-term prognosis than with immediate outcomes. The intensivists' role can be reflected in limited ICU resource utilization.

Kidney-Targeted Cytosolic Delivery of siRNA Using a Small-Sized Mirror DNA Tetrahedron for Enhanced Potency.

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.

Lung-targeted delivery of TGF-ß antisense oligonucleotides to treat pulmonary fibrosis.

Pulmonary fibrosis is a serious respiratory disease, with limited therapeutic options. Since TGF-ß 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 ß-defensin 23 (DhBD23). Antisense oligonucleotides targeting TGF-ß 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.

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.

While siRNA is a potent therapeutic tool that can silence disease-causing mRNA, its in vivo potency can be compromised due to the 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 a DNA tetrahedron, the siRNA can preferentially be accumulated in the 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.

Modulating α-synuclein fibril formation using DNA tetrahedron nanostructures.

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.

Highly tumor-specific DNA nanostructures discovered by in vivo screening of a nucleic acid cage library and their applications in tumor-targeted drug delivery.

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.

Simple and Novel Assay of the Host-Guest Complexation of Homocysteine with Cucurbit[7]uril.

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH2CH2SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.

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.

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.

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.

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.

Self-assembled mirror DNA nanostructures for tumor-specific delivery of anticancer drugs.

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.

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.

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.