Because of the specificity of Watson-Crick base pairing, attempts are now being made to use oligodeoxynucleotides (oligos) in the therapy of human disease. However, for a successful outcome, the oligo must meet at least six criteria: (i) the oligos can be synthesized easily and in bulk; (ii) the oligos must be stable in vivo; (iii) the oligos must be able to enter the target cell; (iv) the oligos must be retained by the target cell; (v) the oligos must be able to interact with their cellular targets; and (vi) the oligos should not interact in a non-sequence-specific manner with other macromolecules. Phosphorothioate oligos are examples of oligos that are being considered for clinical therapeutic trials and meet some, but not all, of these criteria. The potential use of phosphorothioate oligos as inhibitors of viral replication is highlighted.

https://science.sciencemag.org/content/sci/261/5124/1004.full.pdf

Antisense oligonucleotides as therapeutic agents--is the bullet really magical?

Specific regulation of gene expression by antisense, sense and antigene nucleic acids.

Compositions and methods are provided for the treatment and diagnosis of diseases or conditions amenable to treatment through modulation of the synthesis or metabolism of multidrug resistance-associated protein (MRP). In accordance with referred embodiments, oligonucleotides are provided which are specifically hybridizable with nucleic acids encoding MRP. In a preferred embodiment, the oligonucleotide has at least one 2'-methoxyethoxy modification. Methods of preventing the development of multidrug resistance and of improving the efficacy of chemotherapy are also disclosed.

Antisense oligonucleotide modulation of multidrug resistance-associated protein

Oligonucleotides and other macromolecules are provided that have increased nuclease resistance, substituent groups for increasing binding affinity to complementary strand, and sub-sequences of 2′-deoxy-erythro-pentofuranosyl nucleotides that activate RNase H enzyme. Such oligonucleotides and macromolecules are useful for diagnostics and other research purposes, for modulating protein in organisms, and for the diagnosis, detection and treatment of other conditions susceptible to antisense therapeutics.

Gapped 2' modified oligonucleotides

Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties

We have used derivatized antisense oligodeoxynucleotides both in vitro and in vivo specifically to inhibit translation of the activated human oncogene Ha-ras. The oligonucleotides (5'-CCACACCGA-3') were targeted to a region of Ha-ras mRNA including the point mutation G----T at the 12th codon which leads to a Gly----Val substitution in the ras p21 protein. They were linked to an intercalating agent and/or to a hydrophobic tail, both to increase their affinity for their mRNA target and to enhance their uptake by tumor cells. A cell-free translation system was used to demonstrate an RNase H-dependent specific inhibition of activated ras protein synthesis. 50% inhibition was observed at a concentration of 0.5 microM of the most efficient oligonucleotide (5'-substitution with an acridine derivative and 3'-substitution by a dodecanol chain). This inhibitory effect stems from a point mutation-sensitive cleavage of the mRNA and it mirrors the growth inhibition obtained with T24 bladder carcinoma cells, which carry activated Ha-ras. The proliferation of HBL100 cells (non tumorigenic human mammary cell line) which carry two copies of normal Ha-ras was unaffected. This study shows that it is possible to design antisense agents that will inactivate the mutated oncogene but not the protooncogene which is generally essential to cell survival.

Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.

Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine.homopyrimidine sequences by forming local triple helices. Phenanthroline was covalently attached to the 5' end of an 11-mer homopyrimidine oligonucleotide of sequence d(TTTCCTCCTCT). Simian virus 40 DNA, which contains a single target site for this oligonucleotide, was used as a substrate for the phenanthroline-oligonucleotide conjugate. In the presence of copper ions and a reducing agent, a single specific double-strand cleavage site was observed at 20 degrees C by agarose gel electrophoresis. The efficiency of double-strand cleavage was greater than 70% at 20 degrees C and pH 7.4. Secondary cleavage sites were observed when binding of the oligonucleotide to mismatched sequences was allowed to take place at low temperature. The exact location of the cleavage sites was determined by polyacrylamide gel electrophoresis of denatured fragments by using both simian virus 40 DNA and a synthetic DNA fragment containing the target sequence. The asymmetric distribution of the cleavage sites on the two strands revealed that the cleavage reaction took place in the minor groove even though the phenanthroline linker was located in the major groove. Linkers of different lengths were used to tether phenanthroline to the oligonucleotide and their relative efficacies of DNA cleavage were compared. Based on these comparative studies and on model building, it is proposed that the phenanthroline ring carried by the oligonucleotide intercalates from the major groove and that copper chelation locks the complex in place from within the minor groove where the cleavage reaction occurs.

https://www.pnas.org/content/pnas/86/24/9702.full.pdf

Sequence-specific recognition and cleavage of duplex DNA via triple-helix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate.

Sequence-targeted cleavage of single- and double-stranded DNA by oligothymidylates covalently linked to 1,10-phenanthroline.

A homopyrimidine oligonucleotide d(TTTCCTCCTCT) was covalently linked to 1,10-phenanthroline via a 5'-thiophosphate group. In the presence of copper ions and a reducing agent the copper-phenanthroline complex induced cleavage reactions in duplex DNA. The oligonucleotide binds to the major groove of DNA at a homopurine.homopyrimidine sequence, forming a local triple helix. It is oriented parallel to the homopurine strand. Watson-Crick A.T and G.C base pairs are recognized via Hoogsteen-type hydrogen bonding by thymine and protonated cytosine, respectively. The cleavage patterns on opposite strands of duplex DNA at the homopurine.homopyrimidine sequence are asymmetric. They are shifted toward the 3'-side indicating that cleavage takes place from the minor groove even though the oligonucleotide is bound to the major groove. It is therefore suggested that the phenanthroline ring attached to the oligonucleotide intercalates into DNA at the junction between the triple and the double helix and that the copper complex forms in the minor groove where radical reactions leading to strand cleavage occur. The homopyrimidine oligodeoxynucleotide d(TTTCCTCCTCT) tethered to phenanthroline binds to a single site on SV 40 DNA. It cleaves circular and linear SV 40 DNA at this single binding site. Cleavage requires both copper ions and a reducing agent. The unsubstituted oligonucleotide competes with the oligonucleotide-phenanthroline conjugate and prevents site-specific cleavage. These results demonstrate that oligonucleotide-phenanthroline conjugates can be used to induce sequence-specific cleavage of duplex DNA. Such artificial endonucleases could be used, among other things, to map genes on long DNA fragments, to induce site-specific mutations or to block gene expression at the transcriptional level.

M Chassignol

Papers

Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.

Sequence-specific recognition and cleavage of duplex DNA via triple-helix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate.

Sequence-targeted cleavage of single- and double-stranded DNA by oligothymidylates covalently linked to 1,10-phenanthroline.

[Artificial nucleases: specific cleavage of the double helix of DNA by oligonucleotides linked to copper-phenanthroline complex].