Site-Specific Formation of A basic Lesions in DNA

Abstract

A method for the introduction of depurinated lesions in DNA is described, and is based on the formation of a covalent cross-link between an antisense oligonucleotide probe and the target DNA sequence followed by an unexpectedly mild thermal depurination.     

Anti-Influenza Virus Activities of Nicked and Circular Dumbbell RNA/DNA Chimeric Oligonucleotides

Abstract

We have designed a new type of antisense oligonucleotide, containing two hairpin loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem (nicked and circular dumbbell DNA/RNA chimeric oligonucleotides). The reaction of the nicked and circular dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding anti-DNA together with the sense RNA cleavage products. These oligonucleotides were more resistant to exonuclease attack. We also describe the anti-Fluv activities of nicked and circular dumbbell DNMA chimeric oligonucleotides.     

Destabilization of DNA Guanine Quadruplex Structure by Foldback Triplex-Forming Oligodeoxynucleotides

Abstract

An oligodeoxynucleotide designed to bind to a single stranded guanine rich DNA sequence through Watson-Crick followed by Hoogsteen hydrogen bonds was found to destabilize quadruplex structure formed by the target sequence. However, the conventional antisense and antigene oligonucleotides were unable to destabilize the same quadruplex structure.     

Synthesis of a 2-Hydroxy-oxolane Derivative as a New Potential Crosslinking Agent of DNA

Abstract

The design and the synthesis of a new potential crosslinking agent of DNA is reported. It is based on the alkylating properties of a natural toxin (botryodiplodin) and will be further linked to antisense oligonucleotides.     

Preparation of Stereoregulated Antisense Oligodeoxyribonucleoside Phoshorothioate and Interaction with its Complementary DNA And RNA

Abstract

Diastereoisomcric specificity of oligodeoxyribonucleoside phosphorothioate (OPT) in DNA/OPT and RNA/OPT hybrid formation was investigated. The difference in the configuration between RRRR and SSSS was reflected in the conformation and the stability of the DNA/OPT and RNA/OPT hybrids. Therefore, findings of this report rationalize the antisense effect by non-stereoregulated OPT and the difference of diastereoisomerism in susceptibility to RNase H.

     

Conformationally Restrained Chiral PNA Conjugates: Synthesis and DNA Complementation Studies

Abstract

In recent times, PNA (I), a structural mimic of DNA in which the sugar-phosphate backbone is replaced by N-(2-aminoethyl)glycine (aeg) linkage has emerged as a potential antisense therapeutic agent.¹ A major limitation of PNAs from an application perspective is their poor solubility in aqueous medium and being achiral, they bind to cDNA in both parallel (N-PNA/5′-DNA) and antiparallel (N-PNA/3′-DNA) modes. In this connection, we have designed spermine conjugated and conformationally constrained PNA analogues to generate the 4-aminoprolyl backbone (II).² These were synthesised and evaluated for their DNA binding abilities by using UV and CD spectroscopic studies. It is seen that incorporation of one 4-aminoprolyl unit at the N-terminus of a PNA chain not only enhances the inherent binding of PNA to DNA, but also imparts significant bias in parallel and antiparallel binding with cDNA. Conjugation of spermine at C-terminus enhanced the PNA solubility.     

Fast Rna-Rna Annealing in Vitro: Single Cycle Selection Versus Multiple Cycle Selection

Abstract

Fast annealing of complementary RNA in vitro is related to effective antisense RNA-mediated regulation of gene expression and inhibition of viral replication in living cells. Pools of antisense RNA can be selected for species that anneal fast with a given target strand. In this study, we compare the technical and biological advantages and disadvantages of a single round selection assay with extensive multiple cycle selection for fast-annealing antisense RNA species.     

Antisense DNA parameters derived from next-nearest-neighbor analysis of experimental data

Background  

The enumeration of tetrameric and other sequence motifs that are positively or negatively correlated with in vivo antisense DNA effects has been a useful addition to the arsenal of information needed to predict effective targets for antisense DNA control of gene expression. Such retrospective information derived from in vivo cellular experiments characterizes aspects of the sequence dependence of antisense inhibition that are not predicted by nearest-neighbor (NN) thermodynamic parameters derived from in vitro experiments. However, quantitation of the antisense contributions of motifs is problematic, since individual motifs are not isolated from the effects of neighboring nucleotides, and motifs may be overlapping. These problems are circumvented by a next-nearest-neighbor (NNN) analysis of antisense DNA effects in which the overlapping nature of nearest-neighbors is taken into account.     

Making Drugs Out of Oligonucleotides: A Brief Review and Perspective

Abstract

I provide a brief review and perspective thoughts concerning the antisense oligonucleotide, drug discovery paradigm.     

Development of a Rapid Screening System to Test Antisense ODN Modifications and Carriers

Abstract

We developed a rapid screening system, using two reporter genes under the control of the same promoter, to identify the biological activity of modified or/and vectorized antisense oligodeoxynucleotides (ODNs). The ability of a dendrimeric structure and a monocationic cholesterol derivative to enhance ODN cellular uptake was previously investigated by fluorescence analysis. Then, the assay system was validated through investigating the effect of both vectors on antisense ODN efficiency.     

Antisense Properties of Morpholino Oligomers

Abstract

Morpholino oligomers are third generation antisense agents designed to be cost effective, water soluble and resistant to nuclease attack. They show high potency and sequence specificity and follow predictable targeting rules when used in antisense applications in cell culture.     

Identification of a Good C-MYC Antisense Oligodeoxynucleotide Target Site and the Inactivity at This Site of Novel NCH Triplet-Targeting Ribozymes

Abstract

A region of c-myc mRNA was identified which permitted very efficient antisense effects to be achieved in living cells using chimeric methylphosphonate-phosphodiester antisense effectors. Novel inosine—containing ribozymes (which cleave after NCH triplets) were directed to an ACA triplet within this region and delivered into living cells. No ribozyme intracellular activity could be identified. Very low ribozyme function was also observed in in vitro assays using a 1700nt substrate RNA.     

Antisense Knockdown of PKC-α Using LNA-Oligos

Abstract

Full-length and 4 nucleotides truncated Locked Nucleic Acid (LNA) modifications of ISIS 3521 were compared for antisense properties in a cellular assay. ISIS 3521 is a 20-mer phosphorothioate designed to hybridise to human protein kinase C-α (PKC-α) mRNA and is currently submitted to clinical trials against cancer. We report that LNA can potentate this antisense oligo and retain the antisense potential with shorter oligos.     

Antisense Peptides: Tools for Receptor Isolation? Lack of Antisense Msh and Acth to Interact with their Sense Peptides and to Induce Receptor-Specific Antibodies

Abstract

The use of antisense peptides for receptor isolation as proposed by Blalock and his colleages (e.g. TIBTECH 8, 140–144, 1990) was tested for human ACTH as well as α- and β-MSH. We synthesized the corresponding antisense peptides HTCA_h, HSM-α and HSM-β and analyzed them for specific interaction with the sense peptides using several types of binding assay and bioassay. Similarly HTCA_h antibodies were tested for binding to ACTH receptors and ACTH antibodies. All these experiments were negative, i.e. there was no specific interaction between sense and antisense peptides nor between the corresponding antibodies. Receptor binding of the sense peptides was not affected by the antisense peptides or HTCA_h antibodies. Unexpectedly, HTCA_h but not HSM-α or HSM-β was a weak MSH agonist acting through a site independent of the MSH receptor. A detailed analysis of the concept of antisense peptides revealed that the theoretical background of the hypothesis of the ‘molecular recognition theory’ is rather weak, explaining the failure of various attempts to obtain specific receptor antibodies.     

Second Generation Antisense Oligonucleotides—Inhibition of PKC-α and c-raf Kinase Expression by Chimeric Oligonucleotides Incorporating 6″-Substituted Carbocyclic Nucleosides and 2″-O-Ethylene Glycol Substituted Ribonucleosides

Abstract

6′-substituted carbocyclic deoxyribonucleosides and 2′-O-ethylene glycol substituted ribonucleosides have been evaluated as building blocks for antisense oligonucleotides. Within the former class 6′-hydroxy substituted building blocks in combination with internucleoside phosphorothioate linkages have the potential to enhance antisense activity. 2′-O-ethylene glycol substituted ribonucleosides generally allow for the construction of potent antisense oligonucleotides with reduced phosphorothioate content, but differences exist in their effects on biological activity in cell culture in spite of virtually identical effects on RNA-binding affinity. Activity enhancement was most pronounced for a 2′-O-methoxyethyl substituent.     

Specific Inhibition of Hepatitis C Viral Gene Expression by Non-polar (Phenylalkyl)phosphonates

Abstract

Different phenylalkyl backbone modified antisense oligonucleotides complementary to the Hepatitis C virus (HCV) RNA nucleotides 326–342 were synthesized. The lipohilic character of modified oligonucleotides was determined from RP-HPLC retention times. The inhibitory effect of these antisense oligonucleotides on HCV gene expression was analyzed in an in vitro test system.     

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

Enhanced Specificity of Minimally Modified Anti-c-myc Oligonucleotides

Abstract

The sequence-specificity of antisense oligonucleotides (ODN) against c-myc mRNA was tested by Northern blot analysis. Rat smooth muscle cells were treated with antisense or control ODN against c-myc modified by the “minimal protection strategy”. At 0.3 μM concentration the ODN show a very specific reduction in c-myc mRNA levels. Use of the “minimal protection strategy” minimizes nonspecific effects as observed for all-phosphorothioate ODN containing four consecutive guanine residues.     

Natural and Phosphorothioate-Modified Oligodeoxyribonucleotides Exhibit a Non-Random Cellular Distribution

Abstract

Addition of specific antisense oligomers to LTK- cells infected with HSV-1 has been shown to decrease viral production (1,8). We have investigated the cellular components that contain these oligomers and a phosphorothioate derivative by Normarski light microscopy and gel analysis of sucrose gradient cell fractions. Fractionation analysis suggests that these oligomers are distributed throughout the cell in a non-random manner and gel analysis suggest that intact oligomers are not equally distributed in the cytosol, nuclear or membrane component. Information about the cellular location of antisense oligomers should aid in the understanding of their antiviral effect and in the design of more effective oligonucleotide derivatives as potential antiviral agents.