Search

Anti-double-stranded DNA (dsDNA) antibodies induce renal damage in patients with systemic lupus erythematosus by triggering fibrotic processes in kidney cells. However, the precise mechanism underlying penetration of anti-dsDNA immunoglubolin G (IgG) into cells remains unclear. This study was designed to investigate the effect of tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor inducible 14 (Fn14) signaling on anti-dsDNA IgG penetration into cells. Mesangial cells were cultured in vitro, and stimulated with TWEAK and anti-dsDNA IgG. The results revealed that TWEAK dose-dependently enhanced cellular internalization of anti-dsDNA IgG and the expression of high-mobility group box 1 (HMGB1). In addition, TWEAK and anti-dsDNA IgG synthetically downregulate suppressor of cytokine signaling 1, and induce the expression of various fibrotic factors. Furthermore, inhibition of HMGB1 attenuates the enhancement effect of TWEAK on anti-dsDNA IgG internalization. The TWEAK upregulation of HMGB1 involves the nuclear factor-κB and phosphatidylinositide 3-kinase/protein kinase B pathways. Therefore, TWEAK/Fn14 signaling contributes to the penetration of anti-dsDNA IgG and relevant fibrotic processes in mesangial cells., (© 2020 Wiley Periodicals LLC.)

Common fragile sites (CFSs) are genomic loci prone to the formation of breaks or gaps on metaphase chromosomes. They are hotspots for chromosome rearrangements and structural variations, which have been extensively implicated in carcinogenesis, aging, and other pathological processes. Although many CFSs were identified decades ago, a consensus is still lacking for why they are particularly unstable and sensitive to replication perturbations. This is in part due to the lack of high-resolution mapping data for the vast majority of the CFSs, which has hindered mechanistic interrogations. Here, we seek to map human CFSs with high resolution on a genome-wide scale by sequencing the sites of mitotic DNA synthesis (MiDASeq) that are specific for CFSs. We generated a nucleotide-resolution atlas of MiDAS sites (MDSs) that covered most of the known CFSs, and comprehensively analyzed their sequence characteristics and genomic features. Our data on MDSs tallied well with long-standing hypotheses to explain CFS fragility while highlighting the contributions of late replication timing and large transcription units. Notably, the MDSs also encompassed most of the recurrent double-strand break clusters previously identified in mouse neural stem/progenitor cells, thus bridging evolutionarily conserved break points across species. Moreover, MiDAseq provides an important resource that can stimulate future research on CFSs to further unravel the mechanisms and biological relevance underlying these labile genomic regions.

Herein, we report a novel strategy to enhance the antisense activity and the pharmacokinetics of therapeutic oligonucleotides. Through the DNA hybridization chain reaction, DNA hairpins modified with poly(ethylene glycol) (PEG) form a bottlebrush architecture consisting of a double-stranded DNA backbone, PEG side chains, and antisense overhangs. The assembled structure exhibits high PEG density on the surface, which suppresses unwanted interactions between the DNA and proteins (e.g., enzymatic degradation) while allowing the antisense overhangs to hybridize with the mRNA target and thereby deplete target protein expression. We show that these PEGylated bottlebrushes targeting oncogenic KRAS can achieve much higher antisense efficacy compared with unassembled hairpins with or without PEGylation and can inhibit the proliferation of lung cancer cells bearing the G12C mutant KRAS gene. Meanwhile, these structures exhibit elevated blood retention times in vivo due to the biological stealth properties of PEG and the high molecular weight of the overall assembly. Collectively, this self-assembly approach bears the characteristics of a simple, safe, yet highly translatable strategy to improve the biopharmaceutical properties of therapeutic oligonucleotides.

Herein, we report the DNA-mediated self-assembly of bivalent bottlebrush polymers, a process akin to the step-growth polymerization of small molecule monomers. In these "condensation reactions", the polymer serves as a steric guide to limit DNA hybridization in a fixed direction, while the DNA serves as a functional group equivalent, connecting complementary brushes to form well-defined, one-dimensional nanostructures. The polymerization was studied using spectroscopy, microscopy, and scattering techniques and was modeled numerically. The model made predictions of the degree of polymerization and size distribution of the assembled products, and suggested the potential for branching at hybridization junctions, all of which were confirmed experimentally. This study serves as a theoretical basis for the polymer-assembly approach which has the potential to open up new possibilities for suprapolymers with controlled architecture, macromonomer sequence, and end-group functionalities.

The size of the nanocarrier is considered one of the most important issues for its therapeutic effect. Thus, an intelligent nanocarrier with dynamic size has been explored as a promising approach to fulfill the requirements for both efficient accumulation according to the enhanced penetration and retention (EPR) effect and deep penetration into tumor tissue. Herein, structure-switchable triplex DNA was modified on gold nanoparticles (AuNPs) to investigate its potential to modulate the nanoparticle dynamic disassembly process among the tumor microenvironment. We report that the pH-sensitive triplex DNA exhibited outstanding sensitivity and size tunability in triggering the disassembly of AuNP clusters into smaller sizes among the tumor acidic environment, leading to better permeability both in vitro and in vivo. By further combination of the telomerase-sensitive hairpin DNA loaded with chemotherapy drug doxorubicin (DOX), a cancer-specific intracellular drug-release function was also realized, resulting in a precise treatment effect and lower toxicity on normal cells. Through comodification of these two structure-switchable DNA chains on AuNPs and construction of nanoparticle assemblies with proper size, programmed disassembly and drug-release function in tissue and cell level, respectively, were successfully combined and eventually facilitated a highly efficient nanodrug transportation process, from tumor accumulation to deep penetration and precise cancer chemotherapy. The study provided the prospect of utilizing functionalized DNA in optimization of nanocarrier delivery efficiency.

Proteins play a key role in disease diagnosis, and protein discrimination is an important but difficult issue. Here, we report a novel strategy for improving protein discrimination through a facile colorimetric sensor array, which is based on DNA-gold nanoparticle (AuNP) conjugates manipulated by exonuclease I (Exo I). Different proteins exhibit diverse affinities toward the three DNAs, and the DNA-protein binding is resistant to the digestion of Exo I and protects the AuNPs from aggregation in high concentrations of NaCl media, forming distinct response patterns of the array. These response patterns as "fingerprints" can be acquired on the sensor array and then identified by linear discriminant analysis (LDA). The sensor array achieved the correct discrimination of 15 proteins at a 10 nM level in buffer solution and real serum samples. Also, the sensor array had the capability to discriminate individual proteins and the mixtures of them. Remarkably, the practicability of the sensor array was further confirmed by the identification of 35 unknown protein samples with 100% accuracy.

While the solution assembly of amphiphilic copolymers has been studied extensively, the assembly of DNA-containing copolymers is only recently emerging as a promising new area. DNA, a natural hydrophilic biopolymer that is highly predictable in its hybridization characteristics, brings to the field several useful and unique properties including monodispersity, the ability to functionalize in a site-specific manner, and programmability with Watson-Crick base pairing. The inclusion of DNA as a segment in the copolymer not only adds to the current knowledge base in the block copolymer assembly but also creates new modalities of assembly that have already led to novel and technologically useful structures. In this Review, we discuss recent progress in the self-assembly of DNA-containing copolymers, including assemblies driven by hydrophobicity via amphiphilic constructs, programmed assemblies mediated by DNA hybridization, and assemblies involving both of these interactions.

A new proteomic strategy combining functionalized magnetic nanoparticle affinity probes with mass spectrometry was developed to capture and identify proteins specifically responding to 1,2-d(GpG) intrastrand cisplatin-cross-linked DNA, the major DNA lesion caused by cisplatin and thought to induce apoptosis. A 16-mer oligodeoxynucleotide (ODN) duplex and its cisplatin-cross-linked adduct were immobilized on magnetic nanoparticles via click reaction, respectively, to fabricate negative and positive affinity probes which were very stable in cellular protein extracts due to the excellent bio-orthogonality of click chemistry and the inertness of covalent triazole linker. Quantitative mass spectrometry results unambiguously revealed the predominant binding of HMGB1 and HMGB2, the well-established specific binders of 1,2-cisplatin-cross-linked DNA, to the cisplatin-cross-linked ODN, thus validating the accuracy and reliability of our strategy. Furthermore, 5 RNA or single-stranded DNA binding proteins, namely, hnRNP A/B, RRP44, RL30, RL13, and NCL, were demonstrated to recognize specifically the cisplatinated ODN, indicating the significantly unwound ODN duplex by cisplatin cross-linking. In contrast, the binding of a transcription factor TFIIFa to DNA was retarded due to cisplatin damage, implying that the cisplatin lesion stalls DNA transcription. These findings promote understanding in the cellular responses to cisplatin-damaged DNA and inspire further precise elucidation of the action mechanism of cisplatin.

[Ru(bpy)₂dppz] 2+ and [Ru(phen)₂dppz] 2+ as the light switches of the deoxyribose nucleic acid (DNA) molecule have attracted much attention and have become a powerful tool for exploring the structure of the DNA helix. Their interactions have been intensively studied because of the excellent photophysical and photochemical properties of ruthenium compounds. In this perspective, this review describes the recent developments in the interactions of these two classic intercalated compounds with a DNA helix. The mechanism of the molecular light switch effect and the selectivity of these two compounds to different forms of a DNA helix has been discussed. In addition, the specific binding modes between them have been discussed in detail, for a better understanding the mechanism of the light switch and the luminescence difference. Finally, recent studies of single molecule force spectroscopy have also been included so as to precisely interpret the kinetics, equilibrium constants, and the energy landscape during the process of the dynamic assembly of ligands into a single DNA helix.

Alpha-synuclein plays a key role in the pathogenesis of Parkinson's disease (PD). A robust transgenic mouse model has been generated that overexpresses the mutant human A53T alpha-synuclein under the mouse prion protein gene promoter; these mice develop age-dependent motor deficits. Recently, compared to wild-type (WT) littermates, A53T alpha-synuclein mice were reported to display non-motor symptom deficits, e.g., anxiety-like and depressive-like behaviors, odor discrimination and detection impairments, and gastrointestinal dysfunction, at 6 months of age or older. However, the differences between heterozygous and homozygous mice in terms of non-motor symptoms and whether the genomic DNA levels of alpha-synuclein correlate with the symptoms have not yet been elucidated. In the present work, we used littermate WT and heterozygous and homozygous A53T mice that were characterized by a modified genotyping protocol and observed a unilateral decline in the dopamine transporter (DAT) distribution from 3 months to 12 months of age in homozygous mice. We evaluated non-motor symptoms by measuring colon motility, anxiety-like and depressive-like behaviors, and motor coordination. The results showed that homozygous A53T mice exhibited earlier abnormal non-motor symptoms compared to their heterozygous littermates. The severity of impaired colon motility as well as anxiety-like and depressive-like behaviors were correlated with the genomic DNA levels of A53T mutant alpha-synuclein. More noticeable, motor coordination aberrances were also observed in homozygous A53T mice. This study provides direct evidence that the genomic DNA levels of mutant alpha-synuclein correlate with non-motor symptoms in an A53T mouse model, indicating that the genomic DNA levels of mutant alpha-synuclein should be tightly manipulated in PD model studies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

PEGylation of an oligonucleotide using a brush polymer can improve its biopharmaceutical characteristics, including enzymatic stability and biodistribution. Herein, we quantitatively explore the nuclease accessibility of the nucleic acid as a function of "depth" toward the backbone of the brush polymer. It is found that protein accessibility decreases as the nucleotide is located closer to the backbone. Thus, by moving the conjugation point from the terminus of the nucleic acid strand to an internal position, much smaller brushes can be used to achieve the same level of steric shielding. This finding also makes it possible to assess antisense gene regulation efficiency of these brush-DNA conjugates as a function of their nuclease stability.

Nucleic acids are generally regarded as the payload in gene therapy, often requiring a carrier for intracellular delivery. With the recent discovery that spherical nucleic acids enter cells rapidly, we demonstrate that nucleic acids also have the potential to act as a delivery vehicle. Herein, we report an amphiphilic DNA-paclitaxel conjugate, which forms stable micellar nanoparticles in solution. The nucleic acid component acts as both a therapeutic payload for intracellular gene regulation and the delivery vehicle for the drug component. A bioreductively activated, self-immolative disulfide linker is used to tether the drug, allowing free drug to be released upon cell uptake. We found that the DNA-paclitaxel nanostructures enter cells ∼100 times faster than free DNA, exhibit increased stability against nuclease, and show nearly identical cytotoxicity as free drug. These nanostructures allow one to access a gene target and a drug target using only the payloads themselves, bypassing the need for a cocarrier system.

The simultaneous intracellular delivery of multiple types of payloads, such as hydrophobic drugs and nucleic acids, typically requires complex carrier systems. Herein, we demonstrate a self-deliverable form of nucleic acid-drug nanostructure that is composed almost entirely of payload molecules. Upon light activation, the nanostructure sheds the nucleic acid shell, while the core, which consists of prodrug molecules, disintegrates via an irreversible self-immolative process, releasing free drug molecules and small molecule fragments. We demonstrate that the nanostructures exhibit enhanced stability against DNase I compared with free DNA, and that the model drug (camptothecin) released exhibits similar efficacy as free, unmodified drugs toward cancer cells.

Triblock copolymer brushes were functionalized with nucleic acid sequences, which allowed the polymers to connect head-to-tail and form supramolecular nanostructures. Two approaches were designed and implemented, using either a palindromic DNA attached to both ends of the polymer or two different DNA sequences attached regiospecifically. Given appropriate conditions, the DNA-brush conjugates self-assembled to form either nanoworms with length up to several microns or cross-linked networks. This process is analogous to the step-growth polymerization of small molecule monomers.

Interactions between DNA and ruthenium organometallic compounds are studied by using visible light absorption and circular dichroism measurements. A titration technique allowing for the absolute determination of the advancement degree of the complexation, without any assumption about the number of complexation modes is developed. When DNA is in excess, complexation involves intercalation of one of the organometallic compound ligands between DNA base pairs. But, in the high complexation ratio limit, where organometallic compounds are in excess relative to the DNA base pairs, a new mode of interaction is observed, in which the organometallic compound interacts weakly with DNA. The weak interaction mode, moreover, develops when all the DNA intercalation sites are occupied. A regime is reached in which one DNA base pair is linked to more than one organometallic compound.

DNA and poly(N-isopropylacrylamide) are co-assembled onto gold nanoparticles. The DNA sequences can be reversibly exposed or hidden from the polymer surface in response to temperature cues, thereby translating the temperature trigger to the on-off switching of the surface chemistry and function. When exposed by heating (∼30 °C), the DNA rapidly hybridizes to complementary strands, and chain-end biotin groups become readily accessible, while at lower temperatures these activities are largely blocked.

The multiplex system allows the detection of 19 autosomal short tandem repeat (STR) loci [including all Combined DNA Index System (CODIS) STR loci as well as D2S1338, D6S1043, D12S391, D19S433, Penta D and Penta E] plus the sex-determining locus Amelogenin in a single reaction, comprising all STR loci in various commercial kits used in the China national DNA database (NDNAD). Primers are designed so that the amplicons are distributed ranging from 90 base pairs (bp) to 450 bp within a five-dye fluorescent design with the fifth dye reserved for the internal size standard. With 30 cycles, 125 pg to 2 ng DNA template showed optimal profiling result, while robust profiles could also be achieved by adjusting the cycle numbers for the DNA template beyond that optimal DNA input range. Mixture studies showed that 83% and 87% of minor alleles were detected at 9:1 and 1:9 ratios, respectively. When 4 ng of degraded DNA was digested by 2-min DNase and 1 ng undegraded DNA was added to 400 μM haematin, the complete profiles were still observed. Polymerase chain reaction (PCR)-based procedures were examined and optimized including the concentrations of primer set, magnesium and the Taq polymerase as well as volume, cycle number and annealing temperature. In addition, the system has been validated by 3000 bloodstain samples and 35 common case samples in line with the Chinese National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The total probability of identity (TPI) can reach to 8×10(-24), where DNA database can be improved at the level of 10 million DNA profiles or more because the number of expected match is far from one person (4×10(-10)) and can be negligible. Further, our system also demonstrates its good performance in case samples and it will be an ideal tool for forensic DNA typing and databasing with potential application., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

A temperature-responsive pentablock copolymer (PB) was designed to deliver DNA and provide prolonged gene expression by forming a thermogelling release depot after subcutaneous or intratumoral injection. A synthetic barrier gel was developed based on poly(ethylene glycol) diacrylate to enable the released vectors to instantly and continuously transfect cultured cells. The aim of this setup was to provide a simple and controlled in vitro system to mimic tumor matrix to optimize the release system and to study the influence of the continuous and sustained release of the polyplexes on the in vitro transfection. The porosity of the barrier gel was adjusted by addition and removal of Pluronic F127 (PL), and the properties of the gel were characterized by visual inspection and scanning electron microscopy (SEM). Concentrated PB-based vector was administered to the barrier gel and allowed to be released to the buffer. We found that most of feed vector could be released from the barrier gel without unpacking the polymer-DNA complexes. Based on the specific construction of the PB-based vectors, an anti-cancer drug paclitaxel (PTX) was further loaded into the same vector designed for DNA delivery, thus formulating a gene and drug co-delivery system. This system was tested for sustained delivery to human ovarian carcinoma cells SKOV3 using the barrier gel in vitro as a tumor mimic. Transfection efficiency was found to be significantly enhanced by co-delivering PTX, while PTX also showed its effect as an anti-cancer drug to induce cell death. Yet both of the two effects of PTX shown in SKOV3 cells turned out to be dramatically weaker in ARPE-19 cells, a human non-cancerous cell line, which might be related to the nature of the vector itself. Instant release of vectors showed the ability to maintain transfection up to the fourth day, making the gene expression stable at least for the first 5 days. Further study is still needed to improve the duration of effective release., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A six-coordinate copper(II) complex with the ligand 1,3-bis(1-methylbenzimidazol-2-yl)-2-oxopropane (Meobb), with composition [Cu(Meobb)(2)](NO(3))(2)·2CH(3)OH, has been synthesized and characterized by elemental analysis, electrical conductivities, IR, UV-Vis spectral measurements. A study of the electro-chemistry of the copper(II) complex was carried out by using cyclic voltammetry. The molecular structures of the ligand Meobb and the Cu(II) complex were determined by X-ray crystal diffraction. The DNA-binding modes of the ligand and the complex were investigated by electronic absorption titration, ethidium bromide-DNA displacement experiments and viscosity measurements. The experimental evidence indicated the compounds interact with calf thymus DNA through intercalation. Additionally, the Cu(II) complex exhibited potential antioxidant properties in in vitro studies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼4 weeks.

A V-shaped ligand 2,6-bis(2-benzimidazolyl)pyridine (bbp) and its picrate Mn(II) complex have been synthesized and characterized by elemental analysis, molecular conductivities, (1)H NMR, IR, UV-vis spectra and X-ray single crystal diffraction. The crystal structure of [Mn(bbp)(2)](pic)(2)·2DMF is six-coordinated forming a distorted octahedron. The DNA-binding properties of the two compounds were investigated by electronic absorption spectra, fluorescence spectra and viscosity measurements. The experimental results suggest that the two compounds bind to DNA in an intercalation mode. The main reason of the DNA-binding behaviors may be the large coplanar aromatic rings in the V-shaped ligand. As compared to the DNA-binding affinities of the two compounds, the Mn(II) complex is stronger than bbp. This may be due to the V-shaped angle changed., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

A cDNA for human TNF-alpha (615bp) was isolated by means of polymerase chain reaction (PCR) using first strand cDNA from PMA-induced HL-60 cells as template. The result from sequencing the 615 bp cDNA fragment indicated that it corresponded to the entire sequence of mature human TNF coding region. Direct expression of mature human TNF was achieved using a plasmid pHT-1 constructed by ligation of the cDNA and a synthetic DNA. The IPTG-induced bacterial product (hTNF) showed cytotoxicity to mouse L-929 cells. The TNF activity was further identified by neutralization of a specific monoclonal antibody against human TNF-alpha. Approximately 80,000 units of activity were detected per ml of culture at A600 = 2.

Three new full-length cDNAs coding for porcine somatotropin (PST) have been cloned. The sequence data indicate a high degree of polymorphism in the PST sequence. All six known PST sequences are different.