Antisense research and development

Antisense Research and DevelopmentVol. 5, No. 4 Phosphorothioate Oligodeoxynucleotides: Antisense or Anti-Protein?Arthur M. Krieg and C. A. SteinArthur M. KriegSearch for more papers by this author and C. A. SteinSearch for more papers by this authorPublished Online:3 Apr 2009https://doi.org/10.1089/ard.1995.5.241AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Phosphorothioate Oligodeoxynucleotides: Antisense or Anti-Protein?." , 5(4), p. 241FiguresReferencesRelatedDetailsCited bySupportive Oligonucleotide Therapy (SOT) as a Potential Treatment for Viral Infections and Lyme Disease: Preliminary Results3 November 2022 | Infectious Disease Reports, Vol. 14, No. 6Antisense oligonucleotide repress telomerase activity via manipulating alternative splicing or translationBiochemical and Biophysical Research Communications, Vol. 582Recent Advances in Oligonucleotide Therapeutics in Oncology24 March 2021 | International Journal of Molecular Sciences, Vol. 22, No. 7Simultaneous Targeting of Two Master Regulators of Apoptosis with Dual-Action PNA– and DNA–Peptide Conjugates22 June 2020 | Bioconjugate Chemistry, Vol. 31, No. 8Chemical Synthesis and Biological Application of Modified Oligonucleotides31 March 2020 | Bioconjugate Chemistry, Vol. 31, No. 5Interaction of ASOs with PC4 Is Highly Influenced by the Cellular Environment and ASO Chemistry15 May 2020 | Journal of the American Chemical SocietyNucleic acid anticancer agentsHybridization-Independent Effects22 June 2018Nucleic acid binding proteins affect the subcellular distribution of phosphorothioate antisense oligonucleotides9 August 2017 | Nucleic Acids Research, Vol. 45, No. 18Immunostimulatory Properties of Lipid Modified CpG Oligonucleotides24 July 2017 | Molecular Pharmaceutics, Vol. 14, No. 8Antisense Oligonucleotide-Mediated Target-Specific Gene Silencing24 November 2016Pleiotropic action of CpG-ODN on endothelium and macrophages attenuates angiogenesis through distinct pathways25 August 2016 | Scientific Reports, Vol. 6, No. 1Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2′-modifications and enhances antisense activity3 March 2016 | Nucleic Acids Research, Vol. 44, No. 8Synthetic SiRNA Delivery: Progress and ProspectsIn vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity26 October 2015 | Nature Biotechnology, Vol. 33, No. 11Antisense Oligonucleotide–Based Therapeutics13 January 2015Knockdown of αIIb by RNA degradation by delivering deoxyoligonucleotides piggybacked with control vivo-morpholinos into zebrafish thrombocytesBlood Cells, Molecules, and Diseases, Vol. 54, No. 1Therapeutic antisense oligonucleotides against cancer: hurdling to the clinic14 October 2014 | Frontiers in Chemistry, Vol. 2Structure-based programming of lymph-node targeting in molecular vaccines16 February 2014 | Nature, Vol. 507, No. 7493Bioconjugation of siRNA for Site-specific Delivery26 April 2013Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference14 September 2012Improved Performance of Anti-miRNA Oligonucleotides Using a Novel Non-Nucleotide ModifierMolecular Therapy - Nucleic Acids, Vol. 2Theory and limitations to gene therapyLipid Conjugated Oligonucleotides: A Useful Strategy for Delivery15 March 2012 | Bioconjugate Chemistry, Vol. 23, No. 6Liposomal SLA co-incorporated with PO CpG ODNs or PS CpG ODNs induce the same protection against the murine model of leishmaniasisVaccine, Vol. 30, No. 26Evaluation of Novel Adjuvant Eimeria Profilin Complex on Intestinal Host Immune Responses Against Live E. acervulina Challenge InfectionAvian Diseases, Vol. 56, No. 2Advanced morpholino oligomers: A novel approach to antiviral therapyAntiviral Research, Vol. 94, No. 1Induction of potent anti-tumor responses while eliminating systemic side effects via liposome-anchored combinatorial immunotherapyBiomaterials, Vol. 32, No. 22Membrane Anchored Immunostimulatory Oligonucleotides for In Vivo Cell Modification and Localized Immunotherapy17 June 2011 | Angewandte Chemie, Vol. 123, No. 31Membrane Anchored Immunostimulatory Oligonucleotides for In Vivo Cell Modification and Localized Immunotherapy17 June 2011 | Angewandte Chemie International Edition, Vol. 50, No. 31Fluorescence Cross-Correlation Spectroscopy Reveals Mechanistic Insights into the Effect of 2′-O-Methyl Modified siRNAs in Living CellsBiophysical Journal, Vol. 100, No. 12Novel adjuvants and delivery systems for enhancing immune responses induced by immunogens9 January 2014 | Expert Review of Vaccines, Vol. 10, No. 2Chemical Modification of Small Interfering RNA15 February 2011Nanomedicine, Vol. 6, No. 10The use of antisense oligonucleotides in Xenopus oocytesMethods, Vol. 51, No. 1RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic PlatformAnnual Review of Pharmacology and Toxicology, Vol. 50, No. 1Biological Barriers to Therapy with Antisense and siRNA Oligonucleotides8 May 2009 | Molecular Pharmaceutics, Vol. 6, No. 3Brain Tumor Therapy with Antisense Oligonucleotides30 July 2009Chemical Modification of siRNAs for In Vivo Use Mark A. Behlke29 November 2008 | Oligonucleotides, Vol. 18, No. 4Targeting the type 1 insulin-like growth factor receptor as a treatment for cancer15 April 2008 | Expert Opinion on Therapeutic Targets, Vol. 12, No. 5Oligonucleotide antiviral therapeutics: Antisense and RNA interference for highly pathogenic RNA virusesAntiviral Research, Vol. 78, No. 1Antisense Oligonucleotides and siRNA as Specific Inhibitors of Gene Expression: Mechanisms of Action and Therapeutic PotentialA Polysaccharide Carrier to Effectively Deliver Native Phosphodiester CpG DNA to Antigen-Presenting Cells27 May 2007 | Bioconjugate Chemistry, Vol. 18, No. 4Delivery of Antisense Oligonucleotides to Nuclear Telomere RNA by Use of a Complex between Polysaccharide and PolynucleotideBulletin of the Chemical Society of Japan, Vol. 80, No. 6Synthetic Nucleic Acids as Potential Therapeutic Tools for Treatment of Bladder CarcinomaEuropean Urology, Vol. 51, No. 2Progress towards in Vivo Use of siRNAsMolecular Therapy, Vol. 13, No. 4Transferrin-appended β-(1→3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptakee-Journal of Surface Science and Nanotechnology, Vol. 3Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA22 April 2005 | Expert Opinion on Drug Delivery, Vol. 2, No. 1Ten years of antisense inhibition of brain G-protein-coupled receptor functionBrain Research Reviews, Vol. 42, No. 2Specific Inhibition of AQP1 Water Channels in Isolated Rat Intrahepatic Bile Duct Units by Small Interfering RNAsJournal of Biological Chemistry, Vol. 278, No. 8Sequence Dependence of C5-Propynyl-dU,dC-Phosphorothioate Oligonucleotide Inhibition of the Human IGF-I Receptor: mRNA, Protein, and Cell Growth Rhys D. Fogarty, Sandra C. McKean, Paul J. White, Lynne M. Atley, George A. Werther, and Christopher J. Wraight8 July 2004 | Antisense and Nucleic Acid Drug Development, Vol. 12, No. 6The Quest for an Efficacious Antiviral for Respiratory Syncytial Virus24 June 2016 | Antiviral Chemistry and Chemotherapy, Vol. 13, No. 6Complement activation is responsible for acute toxicities in rhesus monkeys treated with a phosphorothioate oligodeoxynucleotideInternational Immunopharmacology, Vol. 2, No. 12Evaluating the Specificity of Antisense Oligonucleotide ConjugatesJournal of Biological Chemistry, Vol. 277, No. 25Development of New Radical Reactions with a Vinylsilyl Group and Their Application to the Synthesis of Branched-Chain Sugar NucleosidesMyc down-regulation induces apoptosis in M14 melanoma cells by increasing p27kip1 levelsOncogene, Vol. 20, No. 22Antisense oligonucleotides in cutaneous therapyPharmacology & Therapeutics, Vol. 90, No. 1Potential therapeutic application of antisense oligonucleotides in the treatment of ocular diseases24 February 2005 | Expert Opinion on Pharmacotherapy, Vol. 2, No. 2The 3′-modified antisense oligos promote faster hydrolysis of the target RNA by RNase H than the natural counterpartTetrahedron, Vol. 57, No. 3EWS Fli-1 Antisense Nanocapsules Inhibits Ewing Sarcoma-Related Tumor in MiceBiochemical and Biophysical Research Communications, Vol. 279, No. 2Selecting optimal antisense reagentsAdvanced Drug Delivery Reviews, Vol. 44, No. 1Electroporation-mediated delivery of 3′-protected phosphodiester oligodeoxynucleotides to the skinJournal of Controlled Release, Vol. 67, No. 2-3Antiproliferative Effects of Steric Blocking Phosphorodiamidate Morpholino Antisense Agents Directed against c-myc ROBERT M. HUDZIAK, JAMES SUMMERTON, DWIGHT D. WELLER, and PATRICK L. IVERSEN30 January 2009 | Antisense and Nucleic Acid Drug Development, Vol. 10, No. 3Highly Nuclease-Resistant Phosphodiester-Type Oligodeoxynucleotides Containing 4‘α- C -Aminoalkylthymidines Form Thermally Stable Duplexes with DNA and RNA. A Candidate for Potent Antisense Molecules2 March 2000 | Journal of the American Chemical Society, Vol. 122, No. 11Gene switching: Analyzing a broad range of mutations using steric block antisense oligonucleotidesA review of issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotidesBiochimica et Biophysica Acta (BBA) - Gene Structure and Expression, Vol. 1489, No. 1Morpholino antisense oligomers: the case for an RNase H-independent structural typeBiochimica et Biophysica Acta (BBA) - Gene Structure and Expression, Vol. 1489, No. 1Cell binding, uptake and cytosolic partition of HIV anti-gag Phosphodiester oligonucleotides 3′-linked to cholesterol derivatives in macrophagesBioorganic & Medicinal Chemistry, Vol. 7, No. 11Comparative inhibitory potential of differently modified antisense oligodeoxynucleotides on hepatitis C virus translationEuropean Journal of Clinical Investigation, Vol. 29, No. 10Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are. Egr-1/NGFI-A-DependentThe American Journal of Pathology, Vol. 155, No. 3Evaluation of Different Types of End-Capping Modifications on the Stability of Oligonucleotides Toward 3′- and 5′-ExonucleasesNucleosides and Nucleotides, Vol. 18, No. 9Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell cultureNucleic Acids Research, Vol. 27, No. 13Antisense Oligonucleotides with Different BackbonesJournal of Biological Chemistry, Vol. 274, No. 31Early Growth Response Factor-1 Induction by Injury Is Triggered by Release and Paracrine Activation by Fibroblast Growth Factor-2The American Journal of Pathology, Vol. 154, No. 3Effect of polyisobutylcyanoacrylate nanoparticles and Lipofectin® loaded with oligonucleotides on cell viability and PKCα neosynthesis in HepG2 cellsBiochimie, Vol. 80, No. 12Sequence Dependency of the Internalization and Distribution of Phosphorothioate Oligonucleotides in Vascular Smooth Muscle CellsBiochemical Pharmacology, Vol. 55, No. 9Nucleosides and Nucleotides. 174. Synthesis of Oligodeoxynucleotides Containing 4‘- C -[2-[[ N -(2-Aminoethyl)carbamoyl]oxy]ethyl]thymidine and Their Thermal Stability and Nuclease-Resistance Properties 114 February 1998 | The Journal of Organic Chemistry, Vol. 63, No. 5Toxicity of Oligodeoxynucleotide Therapeutic AgentsCationic Polyhexylcyanoacrylate Nanoparticles as Carriers for Antisense Oligonucleotides H.-P. ZOBEL, J. KREUTER, D. WERNER, C.R. NOE, G. KÜMEL, and A. ZIMMER30 January 2009 | Antisense and Nucleic Acid Drug Development, Vol. 7, No. 5Diazepam protects against rat hippocampal neuronal cell death induced by antisense oligodeoxynucleotide to GABAA receptor γ2 subunitBrain Research, Vol. 765, No. 1Promise and Problems of Bcl-2 Antisense TherapyJNCI Journal of the National Cancer Institute, Vol. 89, No. 14Morpholino Antisense Oligomers: Design, Preparation, and Properties JAMES SUMMERTON and DWIGHT WELLER30 January 2009 | Antisense and Nucleic Acid Drug Development, Vol. 7, No. 3Morpholino and Phosphorothioate Antisense Oligomers Compared in Cell-Free and In-Cell Systems JAMES SUMMERTON, DAVID STEIN, SUNG BEN HUANG, PAULA MATTHEWS, DWIGHT WELLER, and MICHAEL PARTRIDGE30 January 2009 | Antisense and Nucleic Acid Drug Development, Vol. 7, No. 2β-Actin Messenger RNA Localization and Protein Synthesis Augment Cell Motility24 March 1997 | Journal of Cell Biology, Vol. 136, No. 6Inhibition of carrageenan-induced spinal c-Fos activation by systemically administered c-fos antisense oligodeoxynucleotides may be facilitated by local opening of the blood-spinal cord barrierJournal of Neuroscience Research, Vol. 47, No. 6RNase H-independent antisense activity of oligonucleotide N3'->P5' phosphoramidatesNucleic Acids Research, Vol. 25, No. 4Oligonucleotides and Polynucleotides as Biologically Active CompoundsIn vivo studies with antisense oligonucleotidesTrends in Pharmacological Sciences, Vol. 18, No. 1Sequence-specific antitumor activity of a phosphorothioate oligodeoxyribonucleotide targeted to human C- raf kinase supports an antisense mechanism of action in vivo24 December 1996 | Proceedings of the National Academy of Sciences, Vol. 93, No. 26Specific and nonspecific inhibition of transcription by DNA, PNA, and phosphorothioate promoter analog duplexesBioorganic & Medicinal Chemistry Letters, Vol. 6, No. 23The G-tetrad in antisense targetingTrends in Genetics, Vol. 12, No. 8Inhibition of human telomerase activity by peptide nucleic acidsNature Biotechnology, Vol. 14, No. 5Structures and Motifs Involved in Toll Signaling Volume 5Issue 4Jan 1995 To cite this article:Arthur M. Krieg and C. A. Stein.Phosphorothioate Oligodeoxynucleotides: Antisense or Anti-Protein?.Antisense Research and Development.Jan 1995.241-241.http://doi.org/10.1089/ard.1995.5.241Published in Volume: 5 Issue 4: April 3, 2009PDF download

In our efforts to investigate the biologic role of Ha-ras oncogenes in human melanoma by Ha-ras phosphorothioate antisense oligonucleotides, we observed that antisense, sense, and scrambled control oligonucleotides at a concentration of 10 microM all similarly and strongly inhibited growth of our human melanoma target cell line SK-2 in vitro but without specific decrease of the target protein. Cell numbers with respect to the untreated control were reduced by 84% +/- 4.2% (ISD), 82.9% +/- 3.6%, and 84% +/- 3%, respectively. In vivo studies in a SCID-hu mouse model confirmed these findings. Both antisense and sense control oligonucleotides administered through osmotic pumps significantly (p < 0.006) reduced the mean tumor weight (1.5 g +/- 0.4 g and 1.8 g +/- 0.8 g, respectively) in comparison with saline-treated (5.7 g +/- 0.7 g) or untreated control animals (5.8 g +/- 1.0 g). The vascularity of oligonucleotide-treated tumors was greatly reduced. Clinical signs of oligonucleotide-related toxicity were not observed, and there was no evidence of histopathologic alterations in a variety of mouse tissues. We could demonstrate that the antimelanoma effects can be abrogated in vitro by adding basic fibroblast growth factor (bFGF). In the context of the importance of bFGF in melanocyte biology and angiogenesis, we argue in favor of an interaction between polyanionic phosphorothioate oligonucleotides and bFGF in our melanoma system. These findings stress the notion that phosphorothioate oligonucleotides may be promising antineoplastic lead compounds capable of employing antitumor effects by mechanisms other than specific inhibition of gene expression.

Oligonucleotides are a class of compounds with potential as therapeutics for a variety of clinical applications. Local delivery of oligonucleotides to the arterial wall is a challenging aspect of the development of these therapeutics for restenosis, and herein we report experiments characterizing the uptake and distribution of phosphorothiate oligonucleotides into vascular smooth muscle cells in primary cultures and in rabbit arteries. Primary cultures of smooth muscle cells incubated with rhodamine-oligonucleotides showed uptake only into cytoplasmic vesicles. No nuclear or cytosolic localization was detected. In normal arteries there was no visible tissue or cellular uptake of oligonucleotides after intralumenal administration. However, in balloon-injured arteries there was significant oligonucleotide uptake into the tissue with apparent cytoplasmic delivery to the medial smooth muscle cells, as evinced by intense staining of their nuclei with labeled oligonucleotides. Measurement of FITC-oligonucleotide in artery extracts showed significantly greater uptake in injured, compared with normal arteries. Light and electron microscopic studies demonstrated a correlation between the degree of damage and the amount of uptake. These results demonstrate that oligonucleotides penetrate easily into the arterial wall of balloon-injured arteries and accumulate in the medial smooth muscle cells-the target cells for antirestenosis therapeutics following balloon angioplasty.

The association of urokinase-type plasminogen activator (uPA) with its receptor (uPAR) influences various biologic functions, including cell migration, angiogenesis, differentiation, and wound healing. Expression of uPAR at the mesothelial surface could, therefore, influence cellular responses in the pleural space. We found that a line of cultured human mesothelial cells (MeT5A) expressed specific and saturable binding sites for uPA that increased on stimulation with PMA. Ligand blotting studies showed that the mesothelial receptor is a 50 kD protein similar to that in other cell lines. Binding of active and intact, but not amino terminal or low molecular weight fragment, uPA to mesothelial cells enhanced DNA synthesis and cell proliferation, and antibodies against either the active site of uPA or uPAR abrogated this effect. We reasoned that regulation of uPAR expression could control uPA-induced mitogenesis and tested this hypothesis with antisense oligonucleotides complementary to uPAR mRNA. Phosphorothioate-modified antisense oligonucleotides inhibited uPA-mediated mesothelial cell proliferation in a concentration-dependent manner. These effects were associated with decreased binding of 125I-uPA and reduced expression of the uPAR gene product. The results indicate that uPAR is involved in signal transduction pathways that control uPA-mediated mesothelial cell proliferation, a process implicated in the pathogenesis of mesothelial inflammation and pleural neoplasia. Antisense oligonucleotides to uPAR suppress mesothelial cell mitogenesis in vitro and offer a potential means of regulating the process in vivo.

To clarify the renal disposition characteristics of oligonucleotides at the organ level, the renal handling of model end-capped oligonucleotides, 3'-methoxyethylamine 5'-biotin-decathymidylic acid containing phosphoramidate modifications at 3'- and 5'-terminal internucleoside linkages (T10) and its phosphorothioate (Ts10), were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous outflow and urinary excretion patterns and tissue accumulation of radiolabeled oligonucleotides were evaluated under filtering or nonfiltering conditions. No significant binding to bovine serum albumin (BSA) in the perfusate was observed for T10, whereas more than 90% of Ts10 bound to BSA. The steady-state distribution volume of T10 calculated from the venous outflow pattern was larger than that of inulin, which corresponds to the extracellular volume of the kidney, whereas the distribution volume of Ts10 was larger than that of BSA (the intravascular volume). These results suggested their interaction with the vascular wall. Rapid urinary excretion was observed for T10, similar to inulin used as a marker of golmerular filtration rate. On the other hand, urinary excretion of Ts10 was greatly restricted due to its high binding ability (> 90%) to BSA in the perfusate. A significant amount of T10 and Ts10 was accumulated in the kidney (T10, 1.8% of injected dose; Ts10, 1.3%) compared with inulin (0.2%) and BSA (< 0.1%). The accumulation of these oligonucleotides was ascribed to both tubular reabsorption and uptake from the capillary side. In addition, the uptake of T10 from the capillary side was significantly inhibited by simultaneous injection of dextran sulfate, suggesting that the oligonucleotide was taken up as an anionic molecule. These findings will be useful information for the development of delivery systems for antisense oligonucleotides.

A genetic system for the analysis of antisense and ribozyme mechanisms is a much needed experimental tool, and yeast represent a favorable organism on which to base such a system. We have shown previously that the fission yeast Schizosaccharomyces pombe has potential to satisfy the requirements of such a system. This report describes experiments designed to determine if antisense and ribozyme RNA-mediated gene suppression will be generally applicable to other genes in S. pombe. Antisense and ribozyme RNAs designed to suppress the ade6 gene were expressed at high levels from episomal expression vectors. The ade6 gene was chosen as a target as mutations within the gene confer adenine auxotrophy and a red colony phenotype, and it was expected that antisense or ribozyme RNA-mediated mutant phenocopies would exhibit the same readily detectable phenotype. No phenotypic indication of ade6 suppression was detected in transformed yeast, and ade6 target mRNA was analyzed by primer extension and Northern analysis. Initially, conflicting results were obtained from these techniques, which were determined to be due to duplex formation between antisense and target RNA in vitro. No detectable reduction in the ade6 mRNA levels was found, and it was concluded that the gene was not suppressed by the antisense or ribozyme RNAs tested. These results confirm that in S. pombe as with other organisms, the susceptibility of genes to RNA-mediated suppression may be gene specific and that design of antisense and ribozyme genes will be an empirical process.

Ribozymes catalytically cleave substrate RNA molecules in a sequence-specific manner. Engineered ribozymes can be developed and introduced into tissue culture cells to regulate gene expression and to inhibit viral replication. We have previously reported on the construction of cell lines that constitutively express a single antiviral ribozyme embedded in a lengthy RNA transcript. These cells exhibited a marked reduction in their ability to support viral infection. Here we report the construction of RNA molecules that contain one or two antiviral ribozymes, each specific for a different cleavage site on the genome of the target virus, lymphocytic choriomeningitis virus (LCMV), and each contained in a self-cleavage cassette comprising cis-acting ribozymes designed to release the antiviral molecules from the transcript. In vitro studies showed that both antiviral ribozymes were released properly from the RNAs following cleavage by the flanking ribozymes and that these released ribozymes functioned as expected in cleaving the target virus RNA. These self-cleaving cassettes have been clones into a retroviral vector downstream of, but in the same transcript as, the chloramphenicol acetyltransferase (CAT) gene. Thus, we hoped to employ CAT as a surrogate marker of ribozyme transcription. Stably transformed cell lines were established. Cleavage by the cis-acting ribozymes was incomplete, as assessed by Northern blot analysis and by the ability of transformed cells to produce infectious retroviral particles. Nevertheless, the antiviral ribozyme sequences exerted effects in tissue culture. LCMV RNA levels in ribozyme-expressing cells were suppressed, and infectious virus yields were decreased by up to 95% compared with normal cells and with cells expressing inverted ribozymes. The antiviral effects correlated with CAT levels, but there was no significant difference between cell lines expressing a single ribozymes and those expressing two.

We have successfully used antisense RNA to inhibit replication of the mouse hepatitis virus (MHV) in a cell culture system. MHV is a single-stranded RNA virus of positive polarity. Mouse L2 cells were stably transfected with an antisense construct that targets regions of genes 5 and 6 of the virus, High levels of expression from this construct, which is under control of the human elongation factor 1 alpha promoter, were found. After infection of the antisense cell lines with MHV, replication of the virus was significantly reduced compared with control cells, In a viral plaque assay, smaller plaques were found in the antisense cell lines, In addition, up to a 92% inhibition in the number of viral particles produced in one antisense cell line could be seen. This inhibitory effect decreased at longer (>16 hour) infection times. It was possible to both increase the amount of inhibition and prolong the inhibitory effect by reducing the multiplicity of infection. Our results suggest that antisense RNA may be an effective tool to slow down progression of MHV infection in mice.

The mechanisms and intracellular pathways by which many oligonucleotide analogs enter cells to exert the desired antisense effects are not fully understood and remain a matter of debate. In this study, we describe the synthesis of 5'-digoxigenin-labeled phosphorothioate oligonucleotides and show their use to examine intracellular oligonucleotide distribution within Epstein-Barr virus-transformed B cells. Comparison of digoxigenin-labeled and fluorescein-labeled oligonucleotide distribution shows the same intracellular fate, suggesting that digoxigenin modification does not interfere with intracellular routing. Double immunofluorescence studied by conventional fluorescence and confocal microscopy with antibodies to the labeling molecule and to lysosome-associated membrane protein indicate that oligonucleotides mainly accumulate in the lysosomal compartment. Digoxigenin labeling offers an alternative to study oligonucleotide uptake and distribution by immunoelectron microscopy. Two different approaches have been studied: immunogold labeling in heavily fixed and resin-embedded cells and immunogold labeling in lightly fixed and cryoultramicrotomy processed cells. The results confirm the major lysosomal accumulation of digoxigenin-labeled oligonucleotides and demonstrate that the antigenic capacity of digoxigenin is not damaged by any of the procedures used. Therefore, the conjugation of the functionalized digoxigenin molecule at the 5' end of phosphorothioate oligonucleotides provides a new tool in the study of oligonucleotide uptake and intracellular distribution at both cellular and ultrastructural levels.

To better understand the uptake of oligonucleotides into cells, we have studied the labeling of cell surface proteins by an oligonucleotide conjugated to a radiolabeled photoactivatable crosslinker (Denny-Jaffe reagent). When HL60 cells are treated with the conjugate for 2 hours in a medium containing bovine serum albumin (BSA), almost all of the cell-associated label is found in one protein, which we identify as BSA. Cells grown and treated in a serum-free medium do not show this protein, whereas it is plainly seen in cells that are grown in serum-containing medium but then treated in serum-free medium. Overall association of the oligonucleotide with cells is much higher in serum-free medium than in BSA-containing medium, but the oligonucleotide is mostly not protein-associated in the absence of BSA. We conclude that (1) BSA from the medium serves to block overall association of oligonucleotide with cells, and (2) BSA is the main cell surface protein binding oligonucleotides. We discuss the possible role of albumin in endocytic uptake of oligonucleotides in the cell and in the biodistribution of oligonucleotides in vivo.

To evaluate the effect of synthetic DNA oligomers on regulation of bacterial genes in vivo, we tested 63 oligomers of variable length and chemistry for their ability to selectively suppress light production in the bioluminescent marine organism, Vibrio fischeri. Phosphodiester, phosphorothioate, and mixed backbone oligomers were designed to be lux gene targeted or nontargeted (negative) controls. Although significant suppression of luminescence was observed, most notably with the phosphorothioate oligomers, there was no correlation between inhibitory activity and oligomer sequence. The phosphorothioate oligomer that was most potent for inhibition of luminescence in bacterial culture had no effect on the activity of purified luciferase. Mechanisms other than sequence-specific inhibition of gene expression or direct interaction with luciferase are discussed.

CC-1065 and U-71,184 bind and hyperstabilize DNA duplexes, but little is known about their effects on nucleic acid duplexes of different structure. A 20 mer DNA sequence (5'-TTACTTCAGTTATGAGACCA) containing a drug binding sequence (5'-AGTTA) was selected as the target sequence, and this was duplexed with complementary antisense sequences containing phosphodiester (PO), phosphorothioate (PS), and methylphosphonate (MP) bonds. The duplexes containing PO or PS bound 2 CC-1065 molecules per duplex, presumably at both the target site and at a lower affinity site (5'-AGTAA) on the antisense strand. The duplex containing MP bound only 1 CC-1065, and all duplexes bound only 1 U-71,184. Both CC-1065 and U-71,184 bound to 20 mer duplexes comprised of oligo(dA)-oligo(dT) (2.5 and 2 drugs per duplex, respectively) and poly(rA)-oligo(dT) (1 drug per 20 base pairs). CC-1065 also bound to duplexes between the PO- or PS-based antisense structures and a complementary synthetic 20 mer RNA sequence, with about 1 drug per duplex in each case. CC-1065 increased the Tm for the 20 mer DNA duplexes 17 to 29 degrees C, and the corresponding values for U-71,184 ranged from 7 to 19 degrees C. CC-1065 raised the Tm of oligo(dA)-oligo(dT) and poly(rA)-oligo(dT) 29 degrees C. U71,184 increased the Tm for oligo(dA)-oligo(dT) 30 degrees C but did not significantly elevate the Tm for the corresponding RNA-DNA duplex. The results show that CC-1065 and U-71,184 are capable of binding and stabilizing a variety of nucleic acid duplexes. These agents or their analogs may become useful ligands for antisense oligonucleotide applications.

Direct interaction of oligodeoxynucleotides (ODNs) with proteins represents one of the nonantisense-mediated effects of ODNs. Phosphorothioate-capped ODNs have been shown to inhibit directly the in vitro kinase activity of the chronic myelogenous leukemia-associated protein-tyrosine kinase p210bcr-abl. In this study we have determined the efficacy of this aptameric ODN in a cellular system using the K562 chronic myelogenous leukemia-derived cell line. Significant effects upon cellular phosphotyrosine content, as well as cellular growth in soft agar, are observed. These effects are sequence specific and are not mediated through changes in p210bcr-abl protein levels. Additional ODNs are described that also reduce cellular phosphotyrosine levels and inhibit growth in soft agar but do not inhibit p210bcr-abl kinase activity in vitro.

Phosphorothioate oligonucleotides (S-ODNs) have the ability to modulate gene expression selectively and thus have potential therapeutic capabilities. This potential led us to investigate the protein binding characteristics of selected S-ODNs. We evaluated S-ODN interactions with bovine serum albumin (BSA) and human serum albumin (HSA) in vitro. The equilibrium dissociation constants Km for the binding of a 20 mer S-ODN with BSA and HSA range between 1.1-5.2 x 10(-5) and 2.4-3.1 x 10(-4) M, respectively. The Km for an unrelated 15 mer S-ODN binding with HSA ranges between 3.7 and 4.8 x 10(-5) M. Studies with a fluorescently labeled 27 mer S-ODN suggest cooperative binding (Hill slope = 1.67) and/or the presence of secondary binding sites on the S-ODN. HSA or BSA linked to Sepharose was incubated with a 15, 20, or 24 mer S-ODN followed by the addition of selected drugs known to be highly protein bound (nifedipine, warfarin, midazolam, probenecid, indomethacin, and mitoxantrone). Up to 30% of S-ODN was displaced by warfarin in competition binding assays. Conversely, HSA-bound warfarin was incubated with a variety of oligonucleotides, including RNA and genomic dsDNA. Maximum displacement of warfarin-bound HSA was observed following incubation with 5'-cholesterol-conjugated 20 mer S-ODN. In summary, S-ODNs are likely to interact and displace other therapeutic agents that bind to albumin, particularly those binding at site I.

A "phosphorothioate triester method" was investigated for the solution-phase synthesis of phosphorothioate oligonucleosides. Using fully protected 3'-phosphorothiolate thymidine bearing O-cyanoethyl and S-2,4-dichlorobenzyl groups as phosphorothioate protecting groups, decathymidine nonaphosphorothioate was efficiently assembled through a blockwise procedure. Two side reactions occurred during the deprotection steps: breakage of internucleoside linkages (1.8% per linkage) and formation of phosphate diester linkages (0.9%). Substitution of the dichlorobenzyl group by the more labile 4-nitrobenzyl S-protecting group reduced the extent of internucleoside bond breakage by one-half.

Human megakaryocytic tumor cell lines CHRF-288-11 and HEL (human erythroleukemia) were incubated with antisense phosphodiester (PDE) and phosphorothioate (PS) oligodeoxynucleotides directed against the first six codons of the human serglycin proteoglycan gene. As controls, PDE scrambled and PS sense and scrambled sequences and a probe antisense to a 3' portion of the coding sequence were used. Treatment with PDE-ODNs did not alter the core protein content of cell or culture medium proteoglycans. Treatment with all the PS-ODNs resulted in loss of the 31 kD serglycin core protein in the medium, but not the cell-associated proteoglycans, and concomitant appearance of a heavily labeled core protein band at the dye front. This band appears to arise from truncation of the core protein, which leaves the glycosaminoglycan attachment region intact. The higher molecular weight core proteins, which appear to be derived from a betaglycan-like proteoglycan, were not affected by the PDE or PS-ODN treatment. The same effect was seen with or without electroporation, which was used to enhance uptake of the ODNs. Thus treatment of megakaryocytic tumor cells with PS-ODNs appeared to cause a selective degradation of the serglycin core protein in a sequence-independent manner. Degradation most likely occurred intracellularly, because culture supernatants did not degrade exogenously added serglycin proteoglycan, and the presence of superoxide dismutase and catalase in the culture medium during exposure of the cells to the PS-ODNs did not prevent the degradation.

Antisense Research and DevelopmentVol. 5, No. 3 To Cleave or Not To Cleave: Ribozymes and AntisenseTOD M. WOOLFTOD M. WOOLFSearch for more papers by this authorPublished Online:3 Apr 2009https://doi.org/10.1089/ard.1995.5.227AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byTissue-Specific Delivery of Oligonucleotides14 August 2019Towards Improved Oligonucleotide Therapeutics Through Faster Target Binding Kinetics12 September 2017 | Chemistry - A European Journal, Vol. 23, No. 57Hammerhead Ribozymes in Archaeal Genomes: A Computational Hunt13 January 2016 | Interdisciplinary Sciences: Computational Life Sciences, Vol. 9, No. 2Inhibition of miR-21 in glioma cells using catalytic nucleic acids15 April 2016 | Scientific Reports, Vol. 6, No. 1The use of synthetic polymers for delivery of therapeutic antisense oligodeoxynucleotidesBiomaterials, Vol. 23, No. 2Ribozyme-mediated inhibition of caspase-3 activity reduces apoptosis induced by 6-hydroxydopamine in PC12 cellsBrain Research, Vol. 899, No. 1-2In Vivo Expression of Single-Stranded DNA in Mammalian Cells with DNA Enzyme Sequences Targeted to C-raf YIN CHEN, YONG-JIE JI, ROBERT ROXBY, and CHARLES CONRAD30 January 2009 | Antisense and Nucleic Acid Drug Development, Vol. 10, No. 6Antisense PNA tridecamers targeted to the coding region of ha-ras mRNA arrest polypeptide chain elongationJournal of Molecular Biology, Vol. 294, No. 2Enhancing therapeutic glycoprotein production in Chinese hamster ovary cells by metabolic engineering endogenous gene control with antisense DNA and gene targetingGlycobiology, Vol. 9, No. 9Ribozymes as Biotherapeutic Tools for the Modulation of Gene ExpressionInterfering with Gene Function at the RNA Level with Oligozymes—the Development of New Tools and TherapeuticsCationic Polyhexylcyanoacrylate Nanoparticles as Carriers for Antisense Oligonucleotides H.-P. ZOBEL, J. KREUTER, D. WERNER, C.R. NOE, G. KÜMEL, and A. ZIMMER30 January 2009 | Antisense and Nucleic Acid Drug Development, Vol. 7, No. 5Interstrand Crosslinking Reaction in Transplatin-Modified Oligo-2‵-O-Methyl Ribonucleotide-RNA Hybrids CAROLINE COLOMBIER, MARC BOUDVILLAIN, and MARC LENG29 January 2009 | Antisense and Nucleic Acid Drug Development, Vol. 7, No. 4The Sequence-Specific Cleavage of RNA by Artificial Chemical Ribonucleases ROBERT HÄNER and JONATHAN HALL29 January 2009 | Antisense and Nucleic Acid Drug Development, Vol. 7, No. 4Sequence-Specific Cleavage of RNA Using Macrocyclic Lanthanide Complexes Conjugated to Oligonucleotides: A Structure Activity StudyNucleosides and Nucleotides, Vol. 16, No. 7-9The Structure, Function and Application of the Hammerhead RibozymeEuropean Journal of Biochemistry, Vol. 245, No. 1Anti-oncogene Ribozymes for Cancer Gene TherapyComparison of the Antiviral Efficacy of Ribozymes and Antisense RNA Directed Against Bovine Leukemia Virus rex/tax GLENN H. CANTOR, DIANA M. STONE, TERRY F. McELWAIN, and GUY H. PALMER30 January 2009 | Antisense and Nucleic Acid Drug Development, Vol. 6, No. 4 Volume 5Issue 3Jan 1995 To cite this article:TOD M. WOOLF.To Cleave or Not To Cleave: Ribozymes and Antisense.Antisense Research and Development.Jan 1995.227-232.http://doi.org/10.1089/ard.1995.5.227Published in Volume: 5 Issue 3: April 3, 2009PDF download

4'-Thio-beta-D-oligoribonucleotides (12 mer and 16 mer) containing a mixed base sequence were synthesized via the phosphoramidite solid support approach. These RNA analogs showed very good nuclease resistance as compared with wild-type RNA. Furthermore, 4'-thio-beta-D-oligoribonucleotides were shown to hybridize with a complementary DNA or RNA strand to form a duplex or with a DNA hairpin to form a triple helix. 4'-Thio-RNA binds more tightly to its complementary RNA strand than to its complementary DNA strand. A 4'-thio-RNA:RNA duplex is as stable as a 2'-O-methyl-RNA:RNA duplex. 4'-Thio-RNA, however, forms a 4'-thio-RNA:DNA:DNA triplex with a stability similar to the corresponding triplex with all wild-type DNA.

The use of antisense phosphorothioate oligodeoxynucleotides as tools for modulating gene expression represents a novel strategy for designing drugs to treat a variety of diseases. Several factors, including cellular uptake and internalization of the phosphorothioate oligodeoxynucleotide, are important parameters in determining the effectiveness of antisense agents as therapies. We have used cyclodextrin and its analogs as carriers to increase cellular uptake of phosphorothioate oligodeoxynucleotides. The studies were carried out using S-35-labeled and fluorescent-labeled phosphorothioate oligodeoxynucleotide in human T cell leukemia H9 cell line. Cellular uptake of phosphorothioate oligodeoxynucleotide in the presence of cyclodextrin was found to be concentration and time dependent. Using various cyclodextrin analogs, e.g., 2-hydroxypropyl beta-cyclodextrin (HPCD), hydroxyethyl beta-cyclodextrin (HECD), and a mixture of various hydroxypropyl beta-cyclodextrins (Encapsin), we observed increases in phosphorothioate oligodeoxynucleotide uptake, up to twofold to threefold in 48 hours. Confocal microscopy studies confirmed that oligonucleotide was present intracellularly. Cyclodextrin itself was not toxic at the concentration used. Cyclodextrins did not seem to affect the efflux of phosphorothioate oligodeoxynucleotide from cells. Stability of phosphorothioate oligodeoxynucleotide against endogenous cellular nucleases remained unchanged in the presence of cyclodextrins. These studies suggest that cyclodextrin and its analogs might be used successfully as carriers for oligonucleotide and analogs.