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1.
Nucleic Acids Res ; 29(6): 1293-9, 2001 Mar 15.
Article in English | MEDLINE | ID: mdl-11238995

ABSTRACT

Many genes have been described and characterized that have alternative polyadenylation signals at the 3'-end of their pre-mRNAs. Many of these same messages also contain destabilization motifs responsible for rapid degradation of the mRNA. Polyadenylation site selection can thus determine the stability of an mRNA. Fully modified 2'-O:-methoxy ethyl/phosphorothioate oligonucleotides that hybridize to the 3'-most polyadenylation site or signal of E-selectin were able to inhibit polyadenylation at this site and redirect it to one of two upstream cryptic sites. The shorter transcripts produced after antisense treatment have fewer destabilization sequences, increased mRNA stability and altered protein expression. This study demonstrates that antisense oligonucleotides can be successfully employed to redirect polyadenylation. This is the first demonstration of the use of oligonucleotides to increase, rather than decrease, abundance of a message.


Subject(s)
Oligonucleotides/pharmacology , Poly A/genetics , 3' Untranslated Regions/genetics , Blotting, Northern , Cell Line , DNA, Antisense/genetics , DNA, Antisense/pharmacology , E-Selectin/genetics , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Gene Expression Regulation/drug effects , Humans , Oligonucleotides/chemistry , RNA Splicing , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Thionucleotides/chemistry , Transcription, Genetic/drug effects , Tumor Necrosis Factor-alpha/pharmacology
2.
Methods ; 23(2): 191-8, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11181038

ABSTRACT

In the past decade antisense oligonucleotides (ASOs) have proven to be a useful tool for dissection of gene function in molecular cell biology (Koller, E., Gaarde, W. A., and Monia, B. P. (2000) Trends Pharm. Sci., 21, 142-148), and validation of gene targets in animal models (Crooke, S. T. (1998) Biotechnol. Gen. Eng. Rev. 15, 121-157), as well as a means for therapeutic treatment of human diseases (Bennett, C. F. (1999) Exp. Opin. Invest. Drugs 8, 237-253). An important step toward usage of ASOs in the described applications is identification of an active ASO. This article describes the underlying basis and means for achieving this goal in cell culture.


Subject(s)
Genetic Techniques , Oligonucleotides, Antisense/metabolism , Animals , Blotting, Northern , Cell Line , Cells, Cultured , Humans , Oligonucleotide Array Sequence Analysis , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonuclease H/metabolism , Ribonucleases/metabolism
3.
J Comput Aided Mol Des ; 14(6): 593-610, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10921774

ABSTRACT

Binding of the Tat protein to TAR RNA is necessary for viral replication of HIV-1. We screened the Available Chemicals Directory (ACD) to identify ligands to bind to a TAR RNA structure using a four-step docking procedure: rigid docking first, followed by three steps of flexible docking using a pseudobrownian Monte Carlo minimization in torsion angle space with progressively more detailed conformational sampling on a progressively smaller list of top-ranking compounds. To validate the procedure, we successfully docked ligands for five RNA complexes of known structure. For ranking ligands according to binding avidity, an empirical binding free energy function was developed which accounts, in particular, for solvation, isomerization free energy, and changes in conformational entropy. System-specific parameters for the function were derived on a training set of RNA/ligand complexes with known structure and affinity. To validate the free energy function, we screened the entire ACD for ligands for an RNA aptamer which binds L-arginine tightly. The native ligand ranked 17 out of ca. 153,000 compounds screened, i.e., the procedure is able to filter out >99.98% of the database and still retain the native ligand. Screening of the ACD for TAR ligands yielded a high rank for all known TAR ligands contained in the ACD and suggested several other potential TAR ligands. Eight of the highest ranking compounds not previously known to be ligands were assayed for inhibition of the Tat-TAR interaction, and two exhibited a CD50 of ca. 1 microM.


Subject(s)
HIV Long Terminal Repeat , Ligands , RNA, Viral/chemistry , Arginine , Base Sequence , Binding Sites , Computer Simulation , Drug Design , Gene Products, tat/chemistry , Gene Products, tat/metabolism , HIV-1/genetics , HIV-1/physiology , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , Monte Carlo Method , Nucleic Acid Conformation , RNA, Viral/genetics , Reproducibility of Results , Virus Replication , tat Gene Products, Human Immunodeficiency Virus
4.
Nucleic Acids Res ; 28(6): 1340-7, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10684928

ABSTRACT

The secondary and tertiary structures of a mRNA are known to effect hybridization efficiency and potency of antisense oligonucleotides in vitro. Additional factors including oligonucleotide stability and cellular uptake are also thought to contribute to antisense potency in vivo. Each of these factors can be affected by the sequence of the oligonucleotide. Although mRNA structure is presumed to be a critical determinant of antisense activity in cells, to date little direct experimental evidence has addressed the significance of structure. In order to determine the importance of mRNA structure on antisense activity, oligonucleotide target sites were cloned into a luciferase reporter gene along with adjoining sequence to form known structures. This allowed us to study the effect of target secondary structure on oligonucleotide binding in the cellular environment without changing the sequence of the oligonucleotide. Our results show that structure does play a significant role in determining oligonucleotide efficacy in vivo. We also show that potency of oligonucleotides can be improved by altering chemistry to increase affinity for the mRNA target even in a region that is highly structured.


Subject(s)
Nucleic Acid Conformation , Oligoribonucleotides/chemistry , Oligoribonucleotides/metabolism , RNA, Antisense/metabolism , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Animals , Antigens, CD/genetics , B7-2 Antigen , Base Composition , Base Pairing/genetics , Base Sequence , Binding Sites , COS Cells , Genes, Reporter/genetics , Humans , Inhibitory Concentration 50 , Intercellular Adhesion Molecule-1/genetics , Membrane Glycoproteins/genetics , Nucleic Acid Hybridization/genetics , Oligoribonucleotides/genetics , Proto-Oncogene Proteins c-raf/genetics , RNA/genetics , RNA Stability/genetics , RNA, Antisense/chemistry , RNA, Antisense/genetics , RNA, Messenger/genetics , Ribonuclease H/metabolism , Substrate Specificity , Thermodynamics , Transfection
5.
J Med Chem ; 42(22): 4705-13, 1999 Nov 04.
Article in English | MEDLINE | ID: mdl-10579832

ABSTRACT

High-throughput screening of in-house compound libraries led to the discovery of a novel antibacterial agent, compound 1 (MIC: 12-25 microM against S. pyogenes). In an effort to improve the activity of this active compound, a series of 2-substituted quinazolines was synthesized and evaluated in several antibacterial assays. One such compound (22) displayed improved broad-spectrum antibacterial activity against a variety of bacterial strains. This molecule also inhibited transcription/translation of bacterial RNA, suggesting a mechanism for its antibiotic effects. Structure-activity relationship studies of 22 led to the synthesis of another 24 compounds. Although some of these molecules were found to be active in bacterial growth assays, none were as potent as 22. Compound 22 was tested for its ability to cure a systemic K. pneumonia infection in the mouse and displayed moderate effects compared with a control antibiotic, gentamycin.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Benzoates/chemical synthesis , Quinazolines/chemical synthesis , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Benzoates/chemistry , Benzoates/pharmacology , Enterococcus faecalis/drug effects , Escherichia coli/drug effects , Klebsiella Infections/drug therapy , Klebsiella pneumoniae/drug effects , Male , Mice , Mice, Inbred ICR , Protein Biosynthesis/drug effects , Quinazolines/chemistry , Quinazolines/pharmacology , RNA, Bacterial/genetics , Staphylococcus aureus/drug effects , Streptococcus pyogenes/drug effects , Structure-Activity Relationship , Transcription, Genetic/drug effects
7.
Nucleic Acids Res ; 23(15): 3003-8, 1995 Aug 11.
Article in English | MEDLINE | ID: mdl-7659524

ABSTRACT

Peptide nucleic acid (PNA) strand invasion offers an attractive alternative to DNA oligonucleotide directed triplex formation as a potential tool for gene inhibition. Peptide nucleic acid has been shown to interact with duplex DNA in a process which involves strand invasion of the duplex and binding of one of the DNA strands with two PNA oligomers. By blocking the interaction of a transcription factor with 5' regulatory sequences, PNA might specifically down-regulate gene activity. Here we demonstrate that PNA is capable of specifically blocking interaction of the transcription factor NF-kappa B with the IL2-R alpha NF kappa-B binding site in vitro. We further demonstrate that this interaction is sufficient to prevent transcriptional transactivation both in vitro and when transfected into cells in culture.


Subject(s)
DNA/metabolism , NF-kappa B/metabolism , Oligonucleotides, Antisense/metabolism , Peptides/metabolism , Transcriptional Activation , Base Sequence , DNA/biosynthesis , DNA, Recombinant , Down-Regulation , Genes, Reporter/genetics , HeLa Cells , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides, Antisense/chemical synthesis , Promoter Regions, Genetic/genetics , Receptors, Interleukin-2/genetics , Transfection
8.
AIDS Res Hum Retroviruses ; 10(11): 1497-506, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7888204

ABSTRACT

We have previously reported identification of a phosphorothioate oligonucleotide TTGGGGTT (ISIS 5320) as a potent inhibitor of HIV infection in vitro. The oligonucleotide forms a parallel-stranded, tetrameric guanosine quartet (G-quartet) structure that specifically binds to the HIV envelope glycoprotein (gp120) and inhibits both cell-to-cell and virus-to-cell infection at submicromolar concentrations. In the current study we demonstrate that the tetramer inhibits the infection of laboratory-derived isolates of HIV-1 and HIV-2 in a variety of phenotypically distinct, established human cell lines and a panel of biologically diverse clinical isolates in fresh human peripheral blood lymphocytes and macrophages. The compound was also active against all drug-resistant virus isolates tested. In combination with AZT, ISIS 5320 exhibits additive to slightly synergistic anti-HIV activity. Cell-based mechanism of action studies demonstrate that the compound inhibits the binding of infectious virus and virus-infected cells to uninfected target cells by binding to the cationic V3 loop of the envelope glycoprotein. The G-quartet structure is a potential candidate for use in anti-HIV chemotherapy.


Subject(s)
Antiviral Agents/pharmacology , HIV-1/drug effects , HIV-2/drug effects , Oligodeoxyribonucleotides/pharmacology , Thionucleotides/pharmacology , Antiviral Agents/administration & dosage , Antiviral Agents/chemistry , Base Sequence , Cell Fusion/drug effects , Cell Line , Cytopathogenic Effect, Viral/drug effects , HIV Envelope Protein gp120/metabolism , HIV-1/pathogenicity , HIV-1/physiology , HIV-2/pathogenicity , HIV-2/physiology , Humans , Nucleic Acid Conformation , Oligodeoxyribonucleotides/administration & dosage , Oligodeoxyribonucleotides/chemistry , Peptide Fragments/metabolism , Protein Binding/drug effects , Structure-Activity Relationship , Thionucleotides/administration & dosage , Thionucleotides/chemistry , Zidovudine/administration & dosage
9.
Proc Natl Acad Sci U S A ; 91(4): 1356-60, 1994 Feb 15.
Article in English | MEDLINE | ID: mdl-7906414

ABSTRACT

The phosphorothioate oligonucleotide T2G4T2 was identified as an inhibitor of HIV infection in vitro by combinatorial screening of a library of phosphorothioate oligonucleotides that contained all possible octanucleotide sequences. The oligonucleotide forms a parallel-stranded tetrameric guanosine-quartet structure. Tetramer formation and the phosphorothioate backbone are essential for antiviral activity. The tetramer binds to the human immunodeficiency virus envelope protein gp120 at the V3 loop and inhibits both cell-to-cell and virus-to-cell infection.


Subject(s)
Antiviral Agents/pharmacology , Cell Fusion/drug effects , HIV Envelope Protein gp120/metabolism , HIV-1/drug effects , Oligodeoxyribonucleotides/pharmacology , Thionucleotides/pharmacology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/microbiology , Cells, Cultured , Dose-Response Relationship, Drug , HIV-1/growth & development , HIV-1/pathogenicity , Humans , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/metabolism , Thionucleotides/chemical synthesis , Thionucleotides/metabolism , Virulence/drug effects
10.
Nucleic Acids Res ; 21(8): 1853-6, 1993 Apr 25.
Article in English | MEDLINE | ID: mdl-7684128

ABSTRACT

Combinatorial strategies offer the potential to generate and screen extremely large numbers of compounds and to identify individual molecules with a desired binding specificity or pharmacological activity. We describe a combinatorial strategy for oligonucleotides in which the library is generated and screened without using enzymes. Freedom from enzymes enables the use of oligonucleotide analogues. This dramatically extends the scope of both the compounds and the targets that may be screened. We demonstrate the utility of the method by screening 2'-O-Methyl and phosphorothioate oligonucleotide analogue libraries. Compounds have been identified that bind to the activated H-ras mRNA and that have potent antiviral activity against the human herpes simplex virus.


Subject(s)
Drug Design , Genetic Techniques , Oligonucleotides/chemistry , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Base Sequence , Binding Sites , Gene Library , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides/pharmacology , Proto-Oncogene Proteins p21(ras)/genetics , RNA/chemistry , RNA/metabolism , Simplexvirus/drug effects , Simplexvirus/genetics
11.
Science ; 257(5072): 958-61, 1992 Aug 14.
Article in English | MEDLINE | ID: mdl-1502560

ABSTRACT

A pseudo--half-knot can be formed by binding an oligonucleotide asymmetrically to an RNA hairpin loop. This binding motif was used to target the human immunodeficiency virus TAR element, an important viral RNA structure that is the receptor for Tat, the major viral transactivator protein. Oligonucleotides complementary to different halves of the TAR structure bound with greater affinity than molecules designed to bind symmetrically around the hairpin. The pseudo--half-knot--forming oligonucleotides altered the TAR structure so that specific recognition and binding of a Tat-derived peptide was disrupted. This general binding motif may be used to disrupt the structure of regulatory RNA hairpins.


Subject(s)
HIV/genetics , Nucleic Acid Conformation , Oligoribonucleotides/chemistry , RNA, Viral/chemistry , Base Sequence , Binding Sites , DNA, Viral/metabolism , Gene Products, tat/metabolism , Kinetics , Molecular Sequence Data , RNA, Viral/genetics , RNA, Viral/metabolism , tat Gene Products, Human Immunodeficiency Virus
12.
Nucleic Acids Res ; 20(15): 3945-53, 1992 Aug 11.
Article in English | MEDLINE | ID: mdl-1508680

ABSTRACT

The pol gene of all retroviruses is expressed as a gag-pol fusion protein which is proteolytically processed to produce all viral enzymes. In the human immunodeficiency virus (HIV), the gag and pol genes overlap by 241 nucleotides with pol in the -1 phase with respect to gag. The gag-pol fusion is produced via a -1 ribosomal frameshifting event that brings the overlapping, out-of-phase gag and pol genes into translational phase. Frameshifting occurs at a so called 'shift site' 8-10 nucleotides upstream of a hairpin loop which may play a role in the regulation of frameshifting. We have fused this region of HIV-1 to the 5' end of the firefly luciferase reporter gene in order to quantitatively measure ribosomal frameshifting both in cells and by in vitro translation. A series of 2'-O-methyl oligonucleotides was designed to specifically bind the sequences which flank the gag-pol hairpin. Ribosomal frameshifting is enhanced up to 6 fold by those oligonucleotides which bind the area just 3 to the stem. Oligonucleotides which bind 5' to the stem have no effect on frameshift efficiency. In addition, we have constructed a series of fusion genes which mimic the effect of the bound oligonucleotides with intramolecular hairpins. The results suggest that increasing RNA secondary structure downstream of the shift site increases the frequency of ribosomal frameshifting, and that this effect can be mimicked by antisense oligonucleotides.


Subject(s)
Frameshift Mutation/genetics , Fusion Proteins, gag-pol/genetics , HIV-1/genetics , Oligonucleotides, Antisense/genetics , Ribosomes/metabolism , Amino Acid Sequence , Base Sequence , Fusion Proteins, gag-pol/chemistry , Gene Expression Regulation, Viral/genetics , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides, Antisense/metabolism , Plasmids/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
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