Antisense DNA Oligonucleotides Research Articles

Heat shock proteins in the skin

Heat shock proteins (hsp) are expressed in all cells and organisms. Their expression is induced by heat shock (temperatures above 42 degrees C) and other forms of pathophysiological stress. Elevated levels of hsp protect cells from further stress exposure. Hsp are expressed intracellularly. They are highly conserved throughout evolution indicating hsp being necessary for survival under potentially harmful environmental conditions. Hsp are divided into families according to their molecular weight. The majority of hsp function as molecular chaperones. Chaperone function is characterized by binding to other proteins and mediating their folding, transport and interaction with other molecules. In human epidermis hsp are abundantly expressed and have been linked with functions in cell differentiation and photobiology. Recent research has mainly focused on the 27 and 72 kD hsp that are constitutively expressed in human keratinocytes. ultraviolet radiation (UV)-induced cell death and sunburn cell formation can be inhibited by previous heat shock exposure and UV itself can induce hsp expression. The expression of the 27 kD hsp (hsp27) in epidermal keratinocytes in situ and in culture correlates with differentiation. Expression of hsp27 increases simultaneously with keratinocyte differentiation. For that reason, hsp27 is described as a marker of epidermal differentiation. Changes in the expression and inducibility of hsp have been linked with ageing. In the skin, recent data indicate that hsp72 expression remains remarkably stable with intrinsic ageing. In contrast, levels of hsp27 have been found to be elevated in sun-protected aged skin indicating a link between hsp27 expression and age-dependent epidermal alterations. Regulation of hsp can be modified by pharmacological intervention and the development of safe topical and systemic treatments for the prevention of skin damage and disorders of keratinocyte differentiation can be expected for the future.

Regulation of LPS‐stimulated IL‐10 secretion by PKC in macrophages.

IL-10 is a potent anti-inflammatory cytokine produced by a variety of leukocytes including macrophages. It is important to understand the regulation of IL-10 because of its ability to influence the immune response and suppress inflammation. Macrophages stimulated with LPS secrete small amounts of IL-10. However, when combined with FcγR ligation LPS induces the production of large amounts of IL-10 by macrophages. The signaling pathways responsible for LPS-stimulated IL-10 and the positive effect of the FcγR on LPS-stimulated IL-10 production are not well characterized. PKC is activated by LPS and that activation is altered by FcγR ligation in macrophages. This study evaluated the role of PKC in LPS-stimulated IL-10 secretion and the effect of FcγR ligation on LPS-stimulated IL-10 secretion by mouse peritoneal macrophages. Inhibition of PKC with Gö6976 or Rottlerin decreased LPS-stimulated IL-10 secretion and rescued the effects of FcγR ligation. However, depletion of PKC-α and PKC-ε isoforms with antisense DNA oligonucleotides augmented LPS-stimulated IL-10 secretion. Depletion of PKC-δ with antisense had no effect on LPS-stimulated IL-10 secretion and did not alter the influence of FcγR ligation on LPS-stimulated IL-10 secretion. Thus PKC-δ appears to have no role in the regulation of LPS-stimulated IL-10 secretion by mouse macrophages where as PKC-α and -ε may have a negative influence on LPS-stimulated IL-10 secretion. This study was supported by the American Heart Association Grant 50893T.

Protein disulfide isomerase is a central regulator of NADPH oxidase activity

Redox state is a critical factor determining enzyme activity. The protein disulfide isomerase (PDI) belongs to a class of enzymes that maintains proteins in the adequate redox state by isomerising thiols. Vascular NADPH oxidases are important sources of reactive oxygen species (ROS). As NADPH oxidases are likely to be thiol oxidized by the radicals generated by themselves, we hypothesize that the PDI promotes oxidative stress in vascular cells by maintaining NADPH oxidase activity. Overexpression of fluorescence-tagged fusion constructs revealed colocalization of the NADPH oxidase proteins Nox1, Nox2 and Nox4 with endogenous PDI. Moreover, immuno-precipitation of PDI resulted in co-precipitation of Nox 1, 2 and 4 as well as of the p22phox subunit of the NAPDH oxidase. ROS formation in NADPH oxidase-over-expressing HEK cells was reduced by siRNA directed against PDI. Analysis of NADPH oxidase homologues revealed several highly conserved cysteines. Exchange of some of these amino acids to valine or serine resulted in a substantial loss of function. In cultured vascular smooth muscle cells (VSMC) inhibition of PDI using bacitracin or DNA antisense oligonucleotides inhibited NADPH oxidase activity and significantly attenuated angiotensin II-induced radical-mediated activation of AKT. Therefore, PDI via its action on redox-sensitive cysteines is a novel regulator of NADPH oxidases.

Comparative studies of suppression of malignant cancer cell phenotype by antisense oligo DNA and small interfering RNA

One of the distinguishing features of malignant tumor cells is the ability to proliferate in an anchorage-independent manner; methods that effectively suppress this phenotype may be applicable to the therapeutic inhibition of the malignancy of cancers. Interfering RNA is a potentially powerful tool for cancer therapy because of its specificity of target selection and remarkably high efficiency in target mRNA suppression. We studied the use of two knockdown strategies, antisense oligo DNA (AS-ODN) and small interfering RNA (siRNA), and showed how the anchorage-independent proliferation of malignant cells could be blocked efficiently. Anchorage-independent proliferation of rat fibroblasts transformed with v-src was suppressed with only a single 1-microM dose of AS-ODN; similar suppression using siRNA required treatment with 1 nM siRNA every 12 h. With our experimental system, the molecular stability of AS-ODN allowed the use of a simple treatment regimen to control the amount of the target molecule, providing that the treatment dose was sufficiently high. In comparison, siRNA treatment was effective at lower doses, but more frequent treatment was necessary to achieve the same suppression of proliferation.

DNA Drug Design for Cancer Therapy

DNA (antisense and other oligonucleotides) drug design represents a direct genetic approach for cancer treatment. Such an approach takes advantage of mechanisms that activate genes known to confer a growth advantage to neoplastic cells. The ability to block the expression of these genes allows exploration of normal growth regulation. Progress in DNA drug technology has been rapid, and the traditional antisense inhibition of gene expression is now viewed on a genomic scale. This global view has led to a new vision in antisense technology, the elimination of nonspecific and undesirable side effects, and ultimately the generation of more effective and less toxic nucleic acid medicines. Several antisense oligonucleotides are in clinical trials, are well tolerated, and are potentially active therapeutically. DNA drugs are promising molecular medicines for treating human cancer in the near future.

An optimized isolation and labeling platform for accurate microRNA expression profiling

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in both plants and animals. miRNA genes have been implicated in a variety of important biological processes, including development, differentiation, apoptosis, fat metabolism, viral infection, and cancer. Similar to protein-coding messenger RNAs, miRNA expression varies between tissues and developmental states. To acquire a better understanding of global miRNA expression in tissues and cells, we have developed isolation, labeling, and array procedures to measure the relative abundance of all of the known human mature miRNAs. The method relies on rapid isolation of RNA species smaller than ~40 nucleotides (nt), direct and homogenous enzymatic labeling of the mature miRNAs with amine modified ribonucleotides, and hybridization to antisense DNA oligonucleotide probes. A thorough performance study showed that this miRNA microarray system can detect subfemtomole amounts of individual miRNAs from <1 mug of total RNA, with 98% correlation between independent replicates. The system has been applied to compare the global miRNA expression profiles in 26 different normal human tissues. This comprehensive analysis identified miRNAs that are preferentially expressed in one or a few related tissues and revealed that human adult tissues have unique miRNA profiles. This implicates miRNAs as important components of tissue development and differentiation. Taken together, these results emphasize the immense potential of microarrays for sensitive and high-throughput analysis of miRNA expression in normal and disease states.

Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release

Therapeutic agents based on DNA or RNA oligonucleotides (e.g., antisense DNA oligonucleotide, small interfering RNA) require a regulation of their kinetics in cytoplasm to maintain an optimal concentration during the treatment period. In this respect, delivery of functional nanoparticles containing these drugs into cytoplasm has been thought to have a potential for the cytosolic controlled gene release. In this study, we establish a protocol for the encapsulation of nanoparticles into liposome, which is further fused with ultra violet-inactivated Sendai virus to compose fusogenic liposomes. When nanoparticles were encapsulated in conventional liposomes, endocytosis-mediated uptake of nanoparticles was observed. In contrast, numerous amounts of nanoparticles were delivered into the cytoplasm without any cytotoxicity when the particles were encapsulated in fusogenic liposomes. Additionally, fusogenic liposome showed a high ability to deliver nanoparticles containing DNA oligonucleotides into cytoplasm. These results indicate that this combinatorial nanotechnology using fusogenic liposome and nanoparticle is a valuable system for regulating the intracellular pharmacokinetics of gene-based drugs.

Dermal Delivery of Topically Applied Oligonucleotides via Follicular Transport in Mouse Skin

Antisense oligodeoxynucleotides formulated in cream preparations are being examined in the clinic as topical therapy for psoriasis. To produce their intended anti-inflammatory effects, these large anionic molecules must penetrate the stratum corneum and reach the living epidermis and dermis. A topically applied phosphorothioate antisense oligonucleotide targeted to intercellular adhesion molecule-1 recently was shown to modulate cytokine-inducible target gene expression in engrafted human skin. In this study, we examined the route of entry into mouse skin of fluorochrome-tagged or naked second-generation 2'-O-methoxyethyl-modified oligonucleotides that react specifically with an antibody, using topical cream-based formulations. In hairless mouse skin, immunohistochemical analysis and fluorescence microscopy were unable to detect the presence of oligonucleotide in the epidermis or dermis following topical application although immunostaining was associated with the stratum corneum and fluorescein isothiocyanate-labeled oligonucleotide was observed in hair follicles. Kinetic analysis of oligonucleotide topically applied to hair-clipped BALB/c mouse skin showed early follicular localization, diffusion of oligonucleotide from the mid-follicle, and subsequent dermal accumulation. Saline formulation resulted in oligonucleotide remaining within the hair follicle. These results suggest that oligonucleotide penetration in skin involves a follicular route and further, that topical oligonucleotide therapy may be particularly well suited for altering physiology within the hair follicle and related structures.

Smad1 expression and function during mouse embryonic lung branching morphogenesis

Bone morphogenetic protein (BMP) 4 plays very important roles in regulating developmental processes of many organs, including lung. Smad1 is one of the BMP receptor downstream signaling proteins that transduce BMP4 ligand signaling from cell surface to nucleus. The dynamic expression patterns of Smad1 in embryonic mouse lungs were examined using immunohistochemistry. Smad1 protein was predominantly detected in peripheral airway epithelial cells of early embryonic lung tissue [embryonic day 12.5 (E12.5)], whereas Smad1 protein expression in mesenchymal cells increased during mid-late gestation. Many Smad1-positive mesenchymal cells were localized adjacent to large airway epithelial cells and endothelial cells of blood vessels, which colocalized with a molecular marker of smooth muscle cells (alpha-smooth muscle actin). The biological function of Smad1 in early lung branching morphogenesis was then studied in our established E11.5 lung explant culture model. Reduction of endogenous Smad1 expression was achieved by adding a Smad1-specific antisense DNA oligonucleotide, causing approximately 20% reduction of lung epithelial branching. Furthermore, airway epithelial cell proliferation and differentiation were also inhibited when endogenous Smad1 expression was knocked down. Therefore, these data indicate that Smad1, acting as an intracellular BMP signaling pathway component, positively regulates early mouse embryonic lung branching morphogenesis.

Structure‐Switching Signaling Aptamers: Transducing Molecular Recognition into Fluorescence Signaling

AbstractFor Abstract see ChemInform Abstract in Full Text.

507. Down-Regulation of the Epithelial Sodium Channel ENaC through Antisense Oligonucleotides

Sodium absorption across the airway epithelium is increased in cystic fibrosis (CF) patients and may contribute to abnormal water transport and accummulation of sticky sputum, a hallmark of the CF lung disease. Down-regulation of epithelial sodium channels (ENaC) may attenuate the CF lung disease. One approach to decrease ENaC function is the specific down-regulation of its mRNA. Single-stranded DNA antisense oligonucleotides (ssDNA) bind to mRNA sequence-specifically and trigger RNase-H mediated degradation of the ssDNA/mRNA hybrid. However, careful selection of the ssDNA sequence is required to obtain optimal mRNA reduction. Here, we compared five murine ENaC antisense DNAs (20 nucleotides each) for their ability to decrease ENaC mRNA in M1 cells, a murine kidney cell line that expresses ENaC abundantly. It has previously been demonstrated that the alpha subunit of ENaC is the most crucial for channel function and all five ssDNAs were therefore directed against this subunit. First, fluorescently labelled ssDNA was complexed with Lipofectamine 2000 and transfection efficiency determined. Interestingly, the ssDNA rapidly (after 30 min) accumulated in the nucleus of transfected cells and after 6 hrs >90% cells were transfected. The nuclear localisation of the antisense ODNs was consistent with their proposed site of action. Forty-eight hrs after transfection cells were harvested and mRNA was prepared for quantitative RT-PCR using Taqman. The relative degree of aENaC mRNA reduction varied between antisense molecules and ranged from 5 to 60% (n=6, p<0.05). The most efficient ssDNA was used for in vivo transfection. Genzyme lipid 67 (GL67) has been widely used for gene transfer in vivo, but not for antisense applications. We first assessed if GL67 complexed antisense ssDNA efficiently. Gel retardation studies showed that with a 6:8 molar ratio of cationic lipid:nucleotide almost all ssDNA was complexed (n=3). Light-scatter analysis showed that complexes had an average size of 390±24 nm, but varied in size (n=3). Nasal potential difference assays (PD) have been widely used to measure ion transport across murine nasal epithelium. A decrease in ENaC function would result in a drop in baseline PD, because sodium transport is the main component of basal ion transport in the nasal mucosa. Here, we complexed aENaC antisense or control ssDNA to GL67 (80 mg/mouse) and perfused these complexes into the mouse nose over 75 min via a thin plastic catheter. Twenty-four, 48 and 96 hrs after administration the PD was measured in individual cohorts of animals (n=10/group). After the PD measurements all animals were culled and Taqman RT-PCR carried out on nasal mRNA. We did not detect a drop in aENaC mRNA or changes in PD at any time-point. A critical factor for antisense function in the mouse nose is transfection efficiency. In preliminary experiments (n=3) using fluorescently-labelled ODNs we noted that transfection efficiency varied drastically from animal to animal, and will therefore require further optimisation.

Structure‐Switching Signaling Aptamers: Transducing Molecular Recognition into Fluorescence Signaling

The development of aptamer technology considerably broadens the utility of nucleic acids as molecular recognition elements, because it allows the creation of DNA or RNA molecules for binding a wide variety of analytes (targets) with high affinity and specificity. Several recent studies have focused on developing rational design strategies for transducing aptamer-target recognition events into easily detectable signals, so that aptamers can be widely exploited for detection directed applications. We have devised a generalizable strategy for designing nonfluorescent aptamers that can be turned into fluorescence-signaling reporters. The resultant signaling probes are denoted "structure-switching signaling aptamers" as they report target binding by switching structures from DNA/DNA duplex to DNA/target complex. The duplex is formed between a fluorophore-labeled DNA aptamer and an antisense DNA oligonucleotide modified with a quencher (denoted QDNA). In the absence of the target, the aptamer hybridizes with QDNA, bringing the fluorophore into close proximity of the quencher for efficient fluorescence quenching. When this system is exposed to the target, the aptamer switches its binding partner from QDNA to the target. This structure-switching event is coupled to the generation of a fluorescent signal through the departure of QDNA, permitting the real-time monitoring of the aptamer-target recognition. In this article, we discuss the conceptual framework of the structure-switching approach, the essential features of structure-switching signaling aptamers as well as remaining challenges and possible solutions associated with this new methodology.

Screening of functional antidotes of RNA aptamers against bovine thrombin

A specific RNA aptamer (T705) against bovine thrombin had been obtained after seven rounds of SELEX (systematic evolution of ligands by exponential enrichment) selection from a random RNA library previously. In order to further investigate the relationship between the structure and function of this aptamer, three truncated RNA aptamers, T705a, T705b and T705c, were designed according to the secondary structure of T705 RNA. Our results showed that T705c keeping the precise stem–loop structure but lacking most of the stem region sequence of T705 could inhibit clot formation in vitro in the same way as its parental form. We also report here that single-stranded DNA (ssDNA) antisense oligonucleotides, c′ and c′-22, which were complementary to different portions of T705c could act as efficient antidotes reversing the inhibitory activity of T705. It is demonstrated for the first time that ssDNA antisense oligonucleotides are potential antidotes of RNA aptamers and this may be an effective, rapid strategy to find antidotes of RNA aptamers which would be of important usefulness in basic research and drug screening.

Probing alternative foldings of the HIV-1 leader RNA by antisense oligonucleotide scanning arrays.

Scanning arrays of antisense DNA oligonucleotides provide a novel and systematic means to study structural features within an RNA molecule. We used this approach to probe the structure of the untranslated leader of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This 335 nt RNA encodes multiple important replication signals and adopts two mutually exclusive conformations. The poly(A) and the dimer initiation signal (DIS) sequences of the leader RNA are base-paired in the long-distance interaction (LDI) conformation, but both domains form distinct hairpins in the branched multiple hairpins (BMH) conformation. An RNA switch mechanism has been proposed to regulate the activity of the DIS dimerization signal that is masked in one, yet exposed in the other conformation. The two RNA conformations demonstrate discrete differences in the array-based hybridization patterns. LDI shows increased hybridization in the poly(A) region and decreased hybridization in the DIS region when compared with BMH. These results provide additional evidence for the structure models of the two alternative leader RNA conformations. We also found a correlation between the efficiency of oligonucleotide hybridization and the accessibility of the RNA structure as determined by chemical and enzymatic probing in previous studies. The array approach therefore provides a very sensitive method to detect structural differences in related transcripts.

2P112 アンチセンスoligoDNAの結合を指標としたGF-PmRNAの2次構造解析(核酸 A) 構造・物性)

2P112 アンチセンスoligoDNAの結合を指標としたGF-PmRNAの2次構造解析(核酸 A) 構造・物性)

Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in Drosophila and the identification of putative target genes.

Micro-RNAs are a class of small non-coding regulatory RNAs that impair translation by imperfect base pairing to mRNAs. For analysis of their cellular function we injected different miRNA-specific DNA antisense oligonucleotides in Drosophila embryos. In four cases we observed severe interference with normal development, one had a moderate impact and six oligonucleotides did not cause detectable phenotypes. We further used the miR-13a DNA antisense oligonucleotide as a PCR primer on a cDNA library template. In this experimental way we identified nine Drosophila genes, which are characterised by 3' untranslated region motifs that allow imperfect duplex formation with miR-13 or related miRNAs. These genes, which include Sos and Myd88, represent putative targets for miRNA regulation. Mutagenesis of the target motif of two genes followed by transfection in Drosophila Schneider 2 (S2) cells and subsequent reporter gene analysis confirmed the hypothesis that the binding potential of miR-13 is inversely correlated with gene expression.

A comparison of gene repair strategies in cell culture using a lacZ reporter system.

Synthetic oligonucleotides and DNA fragments of less than 1 kilobase (kb) have been shown to cause site-specific genetic alterations in mammalian cells in culture and in vivo. We have used a lacZ reporter gene system to compare the efficiency of episomal and chromosomal gene repair in human embryonic kidney epithelial cells (HEK293), Chinese Hamster Ovary fibroblasts (CHOK1), human bronchial epithelial cells (16HBE), and mouse embryonic stem (ES) cells. The lacZ gene contains a G to A nucleotide change, (Glu to Lys mutation) that abrogates beta-galactosidase activity. We compared the efficiency of different gene repair methods to correct this mutation and restore beta-galactosidase activity. We evaluated PCR-generated double-stranded DNA fragments of 0.52-1.9 kb, single-stranded DNA oligonucleotides of 20, 35, or 80 bases containing internal phosphorothioate links, and a 68 base RNA:DNA oligonucleotide. All of the oligonucleotides and DNA fragments showed some gene repair ability with an episomal plasmid. Short DNA fragments of 0.52 kb or greater gave the highest frequencies of episomal gene repair while single-stranded DNA oligonucleotides gave the highest frequency of chromosomal repair. In the context of a chromosomal target, antisense DNA oligonucleotides gave 5-fold higher frequencies of gene repair than their sense counterparts. The RNA:DNA chimeric oligonucleotide gave little or no gene repair on either a chromosomal or episomal target.

Protein synthesis of eucaryotic cells could be decreased by antisense-DNA of the multi KH domain protein vigilin

Vigilin, a member of the KH protein family, is exceptional among these proteins as it contains 14 KH domains in consecutive order. Vigilin is present in the nucleus and the cytoplasm of all eucaryotic cells studied so far and has apparently high affinity to tRNA and mRNA. There is circumstantial evidence that vigilin expression parallels high translational activity as demonstrated for pancreatic cells in vitro and in vivo as well as for carcinoma cell lines. On a molecular level we have recently demonstrated that vigilin promotes in vitro the export of tRNA from the nucleus to the translational machinery in the cytoplasm and may hence function as an intercompartimental conveyor. In the present study we show that exposure to a vigilin antisense oligo DNA (VAOD) expectedly resulted in a decrease of vigilin-expression, and was concomitant to lower amylase- and trypsin synthesis in freshly isolated pancreatic cells. In addition, carcinoma cells reacted with an increased mortality under exposure to VAOD giving further support for the notion that vigilin participates in cellular life-sustaining processes such as protein translation.

Role of hippocampal α 2A-adrenergic receptor in methamphetamine-induced hyperlocomotion in the mouse

Psychostimulants, such as methamphetamine (METH), induce psychological dependence and we recently suggested that hippocampal α 2A-adrenergic receptor (α 2A-AR) is involved in METH-induced modulation of central nervous systems. The present study shows that pretreatment with yohimbine dose-dependently decreased the ambulatory hyperactivities induced by METH (2 mg/kg) in mice. Moreover, specific knock-down of the hippocampal α 2A-AR with infusion of anti-sense oligo DNA of α 2A-AR significantly suppressed ambulatory activity induced by METH administration. Infusion of sense oligo DNA of α 2A-AR into mouse hippocampus exerted no effects on the ambulatory activity. These observations strongly suggest the involvement of hippocampal α 2A-AR in the regulation of ambulatory activity induced by METH administration.

Cyclin B synthesis is required for sea urchin oocyte maturation

Sea urchins are members of a limited group of animals in which meiotic maturation of oocytes is completed prior to fertilization. This is different from oocytes of most animals such as mammals and amphibians in which fertilization reactivates an arrested meiotic cycle. Using a recently developed technique for in vitro maturation of sea urchin oocytes, we analyzed the role of cyclin B, the regulatory component of maturation-promoting factor, in the control of sea urchin oocyte meiotic induction and progression. Oocytes of the sea urchin Lytechinus variegatus accumulate significant amounts of cyclin B mRNA and protein during oogenesis. We analyzed cyclin B synthetic requirements in oocytes and early embryos by inhibiting cyclin B synthesis with DNA and morpholino antisense oligonucleotides. Cyclin B synthesis is not necessary for the entry of G2-arrested oocytes into meiosis; however, it is required for the proper progression through meiotic divisions. Surprisingly, mature sea urchin eggs contain significant cyclin B protein following meiosis that serves as a maternal store for early cleavage divisions. We also find that cyclin A can functionally substitute for cyclin B in early embryos but not in oocytes. These studies provide a foundation for understanding the mechanism of meiotic maturation independent of the zygotic cell cycle.