Optimized design of antisense oligomers for targeted rRNA depletion.

RNA sequencing (RNA-seq) is extensively used to quantify gene expression transcriptome-wide. Although often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, digestion using RNaseH and antisense DNA oligomers tiling target rRNAs has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA in a 5-min digestion reaction. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by generating rRNA-depletion oligos for Xenopus laevis and for zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to <5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.

Overcoming multidrug resistance in cancer: Recent progress in nanotechnology and new horizons.

Multidrug resistance (MDR), defined as the ability of cancer cells to gain resistance to both conventional and novel chemotherapy agents, is an important barrier in treating malignancies. Initially, it was discovered that cellular pumps dependent on ATP were the cause of resistance to chemotherapy, and further studies have found that other mechanisms such as increased metabolism of drugs, decreased drug entry, and defective apoptotic pathways are involved in this process. MDR has been the focus of numerous initiatives and countless studies have been undertaken to better understand MDR and formulate strategies to overcome its effects. The current review highlights various nano-drug delivery systems including polymeric/solid lipid/mesoporous silica/metal nanoparticles, dendrimers, liposomes, micelles, and nanostructured lipid carriers to overcome the mechanism of MDR. Nanoparticles are novel gateways to enhance the therapeutic efficacy of anticancer agents at the target site of action due to their tumor-targeting abilities, which can limit the unwanted systemic effects of chemotherapy agents and also reduce drug resistance. Additionally, other innovative strategies including RNA interference as a biological process used to inhibit or silence specific gene expression, natural products as MDR modulators with little systemic toxic effects, which interfere with the functions of proteins involved in drug efflux, and physical approaches such as combination of conventional drug administration with thermal/ultrasound/photodynamic strategies are also highlighted.

Targeting Listeria monocytogenes consensus sequence of internalin genes using an antisense molecule.

As an intracellular pathogen, Listeria monocytogenes can enter host cells where it can replicate and escape detection and eradication by the host immune response making the clearance of infection very challenging. Furthermore, with the advent of antimicrobial resistance, the need for alternative targets is inevitable. Internalin proteins are crucial to this bacterium as they contribute to bacterial entry to the systemic circulation. In this study, we targeted a highly conserved region of these proteins by an antisense sequence that was covalently conjugated to the cell penetrating peptides (CPP) to overcome the challenging delivery barriers. Then, we evaluated the efficiency of this construct in vitro. We also assessed the antigenicity, cytotoxicity, and probability of apoptosis induction by this construct. The studied CPP-PNA inhibited bacterial growth and suppressed the mRNA expression of internalins in a dose-dependent manner. In addition, at all studied concentrations, CPP-PNA significantly reduced the invasion rate of L. monocytogenes in the examined cell lines. Moreover, different concentrations of CPP-PNA did not have a significant antigenic, cytotoxic, and apoptotic properties compared to the control. These results suggest the effectiveness of CPP-antisense in targeting the mRNAs of internalins for various research, therapeutic and preventive purposes. However, additional research is required to evaluate the potency, safety, and pharmacokinetics of this compound for the prevention and treatment of listeriosis.

Peptide nucleic acid mediated inhibition of the bacterial signal recognition particle.

We have identified the bacterial signal recognition particle (SRP) as a novel antibacterial target. As a proof of principle, we used an antisense peptide nucleic acid to target a key SRP RNA. The PNA molecules showed efficient inhibition of SRP function and bacterial cell growth, thereby validating our hypothesis.

The RNF146 E3 ubiquitin ligase is required for the control of Wnt signaling and body pattern formation in Xenopus.

The RING finger protein Rnf146 encodes an E3 ubiquitin ligase capable of targeting poly-ADP-ribosylated substrates for proteasomal degradation. Rnf146 has been identified as a critical regulator of Axin1 and thus of Wnt/ß-catenin signaling. However its physiological significance in vertebrate embryonic development remains to be demonstrated. In this study, we take advantages of early Xenopus embryos to demonstrate that Rnf146 is essential for embryonic pattern formation. Depletion of zygotic Rnf146 using a translation blocking morpholino oligo (MO) results in anteriorized development and increased expression the anterior marker gene Otx2, consistent the notion that Rnf146 is a positive regulator of Wnt/ß-catenin signaling through negatively regulating Axin1 expression. This notion is further supported by examination of the role of maternal Rnf146 in the context of Spemann organizer formation and dorsal axis development. Depletion of maternal Rnf146 using an antisense oligodeoxynucleic acid (ODN) leads to ventralized development and diminished expression of organizer genes. Together, we have provided evidence for the first time that Rnf146 is a critical regulator of embryonic pattern formation in vertebrates.

Mitochondria mediate cell membrane repair and contribute to Duchenne muscular dystrophy.

Dystrophin deficiency is the genetic basis for Duchenne muscular dystrophy (DMD), but the cellular basis of progressive myofiber death in DMD is not fully understood. Using two dystrophin-deficient mdx mouse models, we find that the mitochondrial dysfunction is among the earliest cellular deficits of mdx muscles. Mitochondria in dystrophic myofibers also respond poorly to sarcolemmal injury. These mitochondrial deficits reduce the ability of dystrophic muscle cell membranes to repair and are associated with a compensatory increase in dysferlin-mediated membrane repair proteins. Dysferlin deficit in mdx mice further compromises myofiber cell membrane repair and enhances the muscle pathology at an asymptomatic age for dysferlin-deficient mice. Restoring partial dystrophin expression by exon skipping improves mitochondrial function and offers potential to improve myofiber repair. These findings identify that mitochondrial deficit in muscular dystrophy compromises the repair of injured myofibers and show that this repair mechanism is distinct from and complimentary to the dysferlin-mediated repair of injured myofibers.

Knockdown of different influenza A virus subtypes in cell culture by a single antisense oligodeoxyribonucleotide.

Influenza is a heavy socially significant viral infection that affects humans, birds, and wild and domestic animals. The threat of existing and new highly pathogenic subtypes of influenza A virus (IAV) makes it necessary to develop an effective drug that may affect different IAV strains. For this purpose, oligodeoxynucleotides (DNA fragments) attached to titanium dioxide (TiO2) nanoparticles through a polylysine linker, forming TiO2·PL-DNA nanocomposites, that penetrated into cells without transfection agents were used. For the first time, efficient (≥99.9%) suppression of the reproduction of different subtypes of IAV, including highly pathogenic H5N1 and H1N1, was achieved. These results were obtained using the TiO2·PL-DNA nanocomposite bearing a single antisense oligodeoxynucleotide (0.1µM) targeted to the conserved 3'-noncoding region of RNA segment 5, which is common to all tested strains. Very efficient suppression of the reproduction of different subtypes of IAV was probably achieved due to the use of the proposed delivery system for oligonucleotides in the form of the TiO2·PL-DNA nanocomposites. These results indicate the possibility of creating an efficient drug to affect existing and newly emerging pathogenic IAV strains.

Photoregulating RNA digestion using azobenzene linked dumbbell antisense oligodeoxynucleotides.

Introduction of 4,4'-bis(hydroxymethyl)-azobenzene (azo) to dumbbell hairpin oligonucleotides at the loop position was able to reversibly control the stability of the whole hairpin structure via UV or visible light irradiation. Here, we designed and synthesized a series of azobenzene linked dumbbell antisense oligodeoxynucleotides (asODNs) containing two terminal hairpins that are composed of an asODN and a short inhibitory sense strand. Thermal melting studies of these azobenzene linked dumbbell asODNs indicated that efficient trans to cis photoisomerization of azobenzene moieties induced large difference in thermal stability (ΔTm = 12.1-21.3 °C). In addition, photomodulation of their RNA binding abilities and RNA digestion by RNase H was investigated. The trans-azobenzene linked asODNs with the optimized base pairs between asODN strands and inhibitory sense strands could only bind few percentage of the target RNA, while it was able to recover their binding to the target RNA and degrade it by RNase H after light irradiation. Upon optimization, it is promising to use these azobenzene linked asODNs for reversible spatial and temporal regulation of antisense activities based on both steric binding and RNA digestion by RNase H.

Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers.

Antisense oligonucleotides (AONs) hold promise for therapeutic correction of many genetic diseases via exon skipping, and the first AON-based drugs have entered clinical trials for neuromuscular disorders. However, despite advances in AON chemistry and design, systemic use of AONs is limited because of poor tissue uptake, and recent clinical reports confirm that sufficient therapeutic efficacy has not yet been achieved. Here we present a new class of AONs made of tricyclo-DNA (tcDNA), which displays unique pharmacological properties and unprecedented uptake by many tissues after systemic administration. We demonstrate these properties in two mouse models of Duchenne muscular dystrophy (DMD), a neurogenetic disease typically caused by frame-shifting deletions or nonsense mutations in the gene encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, respiratory failure and neurocognitive impairment. Although current naked AONs do not enter the heart or cross the blood-brain barrier to any substantial extent, we show that systemic delivery of tcDNA-AONs promotes a high degree of rescue of dystrophin expression in skeletal muscles, the heart and, to a lesser extent, the brain. Our results demonstrate for the first time a physiological improvement of cardio-respiratory functions and a correction of behavioral features in DMD model mice. This makes tcDNA-AON chemistry particularly attractive as a potential future therapy for patients with DMD and other neuromuscular disorders or with other diseases that are eligible for exon-skipping approaches requiring whole-body treatment.

Involvement of connexin43 in the acute hyperosmotic stimulus­induced synthesis and release of vasopressin in the supraoptic nucleus of rats.

There is evidence that astroglial connexin43 (Cx43) in the supraoptic nucleus (SON) is required for the hyperosmolarity­induced increase in Fos protein expression in magnocellular neurosecretory cells (MNCs). In the present study, the role of astroglial Cx43 in the synthesis and release of vasopressin (VP) by MNCs in the SON subjected to hyperosmotic stimulus was examined. The results revealed that the VP levels in the SON and plasma were increased following acute hyperosmotic stimulus. Treatment of MNCs with Cx43­specific antisense oligodeoxynucleotides (ASODN), which temporarily reduced Cx43 protein production, limited the VP synthesis and release induced by a hyperosmotic stimulus. Similarly, the addition of gap junction and Cx43 hemichannel blockers also attenuated the VP synthesis and release induced by an acute hyperosmotic stimulus. A high extracellular [Ca2+]([Ca2+]o) has been demonstrated to reduce the gap junction activity or opening probability of Cx54 hemichannels. Notably, it was identified that high [Ca2+]o attenuated the VP synthesis and release induced by acute hyperosmotic stimulus, while low [Ca2+]o had a weak or no effect. These results suggested that Cx43 participates in the VP synthesis and release induced by hyperosmotic stimulation in the SON.

A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition.

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time-consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co-introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic-resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high-throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant-specific DNA herbicides.

Protein interactions in Xenopus germ plasm RNP particles.

Hermes is an RNA-binding protein that we have previously reported to be found in the ribonucleoprotein (RNP) particles of Xenopus germ plasm, where it is associated with various RNAs, including that encoding the germ line determinant Nanos1. To further define the composition of these RNPs, we performed a screen for Hermes-binding partners using the yeast two-hybrid system. We have identified and validated four proteins that interact with Hermes in germ plasm: two isoforms of Xvelo1 (a homologue of zebrafish Bucky ball) and Rbm24b and Rbm42b, both RNA-binding proteins containing the RRM motif. GFP-Xvelo fusion proteins and their endogenous counterparts, identified with antisera, were found to localize with Hermes in the germ plasm particles of large oocytes and eggs. Only the larger Xvelo isoform was naturally found in the Balbiani body of previtellogenic oocytes. Bimolecular fluorescence complementation (BiFC) experiments confirmed that Hermes and the Xvelo variants interact in germ plasm, as do Rbm24b and 42b. Depletion of the shorter Xvelo variant with antisense oligonucleotides caused a decrease in the size of germ plasm aggregates and loosening of associated mitochondria from these structures. This suggests that the short Xvelo variant, or less likely its RNA, has a role in organizing and maintaining the integrity of germ plasm in Xenopus oocytes. While GFP fusion proteins for Rbm24b and 42b did not localize into germ plasm as specifically as Hermes or Xvelo, BiFC analysis indicated that both interact with Hermes in germ plasm RNPs. They are very stable in the face of RNA depletion, but additive effects of combinations of antisense oligos suggest they may have a role in germ plasm structure and may influence the ability of Hermes protein to effectively enter RNP particles.

Penicillin facilitates the entry of antisense constructs into Streptococcus mutans.

Antisense oligonucleotides (AS-ODN) target genes in a sequence-specific manner inhibit gene function and have potential use as antimicrobial agents. Cell barriers, such as peptidoglycan, cell surface proteins and lipopolysaccharide membranes, prevent delivery of AS-ODN into the bacterial cell, limiting their use as an effective treatment option. The ß-lactam antibiotic penicillin was examined for its ability to deliver phosphorothioate oligodeoxyribonucleotides (PS-ODNs) and γ(32) P-ODN into Streptococcus mutans OMZ175. Treatment of lag-phase S. mutans OMZ175 cells with penicillin and FBA (PS-ODN targeting the fructose-biphosphate aldolase gene), resulted in prolonged suppression of growth (> 24 h) and fba expression (656.9 ± 194.4-fold decrease at 5 h). Suppression of both cell growth and fba expression corresponded with a greater amount of γ(32) P-ODN becoming cell associated, with a maximum γ(32) P-ODN concentration per cell achieved 5 h after penicillin treatment (6.50 ± 1.39 × 10(8) molecules per CFU). This study confirms that for S. mutans OMZ175, the peptidoglycan layer acts as a major barrier preventing AS-ODN penetration and suggests that the use of agents such as penicillin that interfere with peptidoglycan integrity can significantly increase the uptake of PS-ODN by these cells.

Antisense oligodeoxynucleotide inhibition as an alternative and convenient method for gene function analysis in pollen tubes.

Antisense oligodeoxynucleotide (A-ODN) inhibition works well in animal cells. However, there have been few successful examples to date of its application in plants, and more specifically whether the technique can be used in pollen tubes as a model of plant cell growth. NtGNL1 plays an important role in pollen tube development and was thus selected as an indicator to assess the biological effects of A-ODN. An A-ODN inhibition technique was used to down-regulate NtGNL1 expression in tobacco pollen tubes and showed that A-ODNs could quickly enter pollen tubes through the thick wall and cell membrane and effectively block NtGNL1 expression. Phenotype analysis revealed that the down-regulation of NtGNL1 by A-ODNs resulted in abnormalities in endocytosis and subsequent vesicle trafficking, similar to the phenotypes of pollen tubes treated with NtGNL1 RNAi. This investigation confirmed that A-ODNs could specifically inhibit target gene expression, and furthermore demonstrated that A-ODN functioned in a concentration- and duration-dependent manner, because A-ODNs could be degraded when incubated with pollen tubes. Thus, the A-ODN technique was successfully used for gene function analysis in pollen tubes and appears to be an alternative and convenient technique when the in vitro pollen tube is used as the study model. This technique will greatly facilitate investigations on the molecular mechanism(s) underlying pollen tube growth.

Effect of antisense oligodeoxynucleotide targeted against NF-κB/P65 on cell proliferation and tumorigenesis of gastric cancer.

To study the inhibitory effect of nuclear transcription factor-kappa B(NF-κB) antisense oligodeoxynucleotide(ASODN) on the growth and tumorgenesis of human gastric cancer. We synthesized and transfected the ASODN of NF-κB/P65 to gastric cancer cell line. The effect of ASODN of NF-κB/P65 on the proliferation of gastric cancer cells was measured by MTT method. The subcutaneous xenograft model of human gastric cancer was established in nude mice, and the tumor growth curve was observed. The cell proliferation was significantly inhibited in P65 ASODN-transfected group in vitro (P<0.05). In vivo, tumor formation test showed that the tumor volume in nude mice in ASODN group was obviously smaller than in other groups (P<0.05); the apoptosis index (AI) was significantly higher (P<0.001). Simultaneously, MVD in ASODN group was markedly lower than in other groups (P<0.01). NF-κB could be used as a new biological therapeutic target of gastric cancer.

Static micromixer-coaxial electrospray synthesis of theranostic lipoplexes.

Theranostic lipoplexes are an integrated nanotherapeutic system with diagnostic imaging capability and therapeutic functions. They hold great promise to improve current cancer treatments; however, producing uniform theranostic lipoplexes with multiple components in a reproducible manner is a highly challenging task. Conventional methods, such as bulk mixing, are not able to achieve this goal because of their macroscale and random nature. Here we report a novel technique, called the static micromixer-coaxial electrospray (MCE), to synthesize theranostic lipoplexes in a single step with high reproducibility. In this work, quantum dots (QD605) and Cy5-labeled antisense oligodeoxynucleotides (Cy5-G3139) were chosen as the model imaging reagent and therapeutic drug, respectively. Compared with bulk mixing, QD605/Cy5-G3139-loaded lipoplexes produced by MCE were highly uniform with polydispersity of 0.024 ± 0.006 and mean diameter by volume of 194 ± 15 nm. MCE also showed higher encapsulation efficiency of QD605 and Cy5-G3139. QD605 and Cy5 also formed the Förster resonance energy transfer pair, and thus the cellular uptake and intracellular fate of theranostic lipoplexes could be visualized by flow cytometry and confocal microscopy. The lipoplexes were efficiently delivered to A549 cells (non-small cell lung cancer cell line) and down-regulated the Bcl-2 gene expression by 48 ± 6%.

Suppression of microRNA-96 expression inhibits the invasion of hepatocellular carcinoma cells.

MicroRNA-96 (miR-96) expression is dysregulated in certain types of cancers. However, the role of miR-96 in hepatocellular carcinoma (HCC) invasion and metastasis remains elusive. miR-96 expression was investigated in a number of HCC cell lines by quantitative reverse transcription­polymerase chain reaction (RT-PCR). Cell invasive ability of metastatic HCCLM6 cells transfected with anti-miR-96 oligonucleotides or miR-96 mimic was determined by Matrigel invasion assay in vitro. In addition, metastasis-associated protein, osteopontin, was evaluated by Western blotting. miR-96 expression was significantly increased in highly metastatic HCCLM6 cells. Decreasing miR-96 expression with anti-miR-96 oligonucleotides led to reduced migration and invasion of HCCLM6 cells in vitro. In particular, down-regulation of miR-96 decreased osteopontin expression in HCCLM6 cells. Suppression of miR-96 expression inhibits the invasion of HCC cells, suggesting that miR-96 may be a therapeutic target for inhibiting HCC invasion and metastasis.

Antisense oligodeoxynucleotide targeting HER2 mRNA sensitized docetaxel in breast cancer treatment.

CONTEXT: Human epidermal growth factor receptor 2 (HER2) is one of the oncogenes closely associated with the development and prognosis of breast carcinoma. Down-regulation of HER2 mRNA by antisense oligodeoxynucleotide (ASO) HER2 has been suggested to be a feasible treatment for patients with breast carcinoma. OBJECTIVE: The antitumor effects of ASO HA6722 were investigated in vitro and in vivo. MATERIALS AND METHODS: In this study, SK-BR-3, a HER2-overexpressing breast carcinoma cell line, was used as the model for in vitro experiments. Inhibitory effects of the ASO HA6722 were detected by methyl-thiazoldiphenyl tetrazolium (MTT) assay. Meanwhile, HER2 mRNA levels were monitored by reverse transcription polymerase chain reaction (RT-PCR). The in vivo antitumor effects were evaluated in nude mice xenograft model. RESULTS: Our results showed that HA6722 alone could inhibit the growth of SK-BR-3 cells in a dose-dependent manner with the IC(50) value of 41.8 ± 8.1 nM. In addition, the antitumor effect of docetaxel (TXT) could be sensitized by low dose of HA6722 both in vitro and in vivo, suggesting that ASO HA6722 could inhibit the growth of breast cancer cells and enhance the cytotoxic effects of TXT. DISCUSSION AND CONCLUSION: The combination treatment of TXT and HA6722 could be a more effective approach for breast cancer treatment. The future study should focus on the antitumor effect in other models.

Uptake and intracellular fate of multifunctional nanoparticles: a comparison between lipoplexes and polyplexes via quantum dot mediated Förster resonance energy transfer.

Lipoplexes and polyplexes represent the two major nanocarrier systems for nucleic acid delivery. Previous studies examining their uptake and intracellular unpacking rely on organic fluorophores fraught with low signal intensity and photobleaching. In this work quantum dot mediated Förster resonance energy transfer (QD-FRET) was first used to study and compare the cellular uptake and the intracellular fate of oligodeoxynucelotide (ODN)-based lipoplexes and polyplexes. QD605-amine and Cy5-labeled ODN (Cy5-GTI2040) were chosen as the FRET pair. By adjusting the lipid/ODN ratio of lipoplexes and the nitrogen/phosphate (N/P) ratio of polyplexes, lipoplexes and polyplexes with comparable physical properties were produced. The biological activities of dual-labeled lipoplexes and polyplexes remained unaltered compared to their unlabeled counterparts as evidenced by their comparable antisense activities against protein R2 in KB cells. Flow cytometry and confocal microscopy revealed similar pattern of uptake for these two types of nanoparticles, although polyplexes had a higher dissociation rate than lipoplexes in KB cells. We demonstrate that QD-FRET is a sensitive tool to study the uptake and intracellular unpacking of lipoplexes and polyplexes, which may help optimize their formulations for various theranostics applications.