Bernard Rayner

Oligodeoxynucleotide stability in subcellular extracts and culture media

Degradation of a synthetic alpha-oligodeoxynucleotide was studied in order to compare its survival with naturally occurring beta-oligodeoxynucleotides in five systems used for antisense hybridization arrest experiments. In contrast to beta-oligodeoxynucleotides, alpha-oligodeoxynucleotides were not detectably degraded over 24 h at 37 degrees C in HeLa cell postmitochondrial cytoplasmic extract or RPMI 1640 with 10% fetal bovine serum, and showed significant survival after 24 h at 37 degrees C in rabbit reticulocyte lysate, fetal bovine serum and human serum.

Antiviral activity of conjugates between poly(l-lysine) and synthetic oligodeoxyribonucleotides ☆

Short (14 to 20-mer range) synthetic oligodeoxyribonucleotides (oligos) allow to modulate specifically viral or cellular gene expression at various stages thus providing a versatile tool for fundamental studies and a rational approach to antiviral chemotherapy. Several problems, such as metabolic stability and efficient cell internalization of oligos, still limit this approach appreciably, as briefly discussed here. We demonstrate here that the conjugation of 15-mer (beta)-anomeric oligos to poly(L-lysine) allows a specific protection of various cell lines against vesicular stomatitis virus infection at concentrations lower than 1 microM. This can be achieved with oligos complementary to the viral N-protein mRNA initiation site or to viral intergenic sequences, i.e., to untranscribed regions. No antiviral activity can be obtained with (alpha)-anomeric oligos directed against the same targets, although such analogues are much more resistant to nuclease degradation and form stable hybrids, at least in cell-free experiments.

Mutagenic properties of a unique abasic site in mammalian cells

The mutagenic properties of a true unique abasic site located opposite a guanine residue were studied. An oligonucleotide containing a chemically-produced abasic site was inserted into a shuttle vector able to replicate both in simian cells and in bacteria. Plasmid DNA was rescued from simian cells and screened in bacteria by differential hybridization with a labelled oligonucleotide probe. Mutations were easily detected and sequenced. Results showed that opposite a guanine the abasic site was error free repaired or replicated by mammalian cells with an efficiency of 99%. Point mutations occurred at a frequency of approximately 1% in control host cells and at more than 3% in UV-pre-irradiated host cells. Adenine, cytosine or thymine were found to have been inserted opposite the abasic site. No preferential insertion for a particular base was observed in contrast to that reported in bacteria.

THE PROOLIGONUCLEOTIDE APPROACH. I: ESTERASE-MEDIATED REVERSIBILITY OF DITHYMIDINE S-ALKYL-PHOSPHOROTHIOLATES TO DITHYMIDINE PHOSPHOROTHIOATES

Abstract Alkylation of dithymidine phosphorothioate and phosphorodithioate with various iodoalkyl acylates afforded the corresponding uncharged S-alkyl phosphoromono- and di-thioates respectively. Upon incubation of these triesters in CEM cell extracts, the bioreversible alkyl acylate masking groups were selectively and rapidly removed by carboxyesterases present in the milieu, yielding the starting dinucleoside diesters.

Studies on the Δδ criterion for determining the anomeric configuration of ribofuranosyl nucleosides

Abstract Studies on the origin of the criterion for determination of the anomeric configuration of D -ribofuranosyl nucleosides, based on the differences in the chemical shift (Δδ) between the Me signals of the corresponding 2′,3′- O -isopropylidene derivatives, is discussed. Proof of the anisotropic influence of the aglycon group on the chemical shifts of the Me groups is given by a comparison of the chemical shifts of anomeric pairs of nucleosides having a reduced aglycon group with those of the non-reduced heterocycle. Therefore, the criterion appears to be limited to ribofuranosyl compounds having an unsaturated heterocyclic aglycon.

Sugar modified oligonucleotides. I. Carbo-oligodeoxynucleotides as potential antisense agents.

For the first time, carbo-oligodeoxynucleotides, namely c-dT4 and c-dT12, have been synthesized. As compared to the natural oligomers these carbo-oligodeoxynucleotides are at least 5 times more stable toward enzymatic degradation and bind more strongly to complementary DNA. These preliminary data indicate that such oligomers fulfill the requirements to be considered as potential antisense agents.

Photolabile linker for the solid-phase synthesis of base-sensitive oligonucleotides

Abstract o-Nitrophenyl-1,3-propanediol was covalently linked to long chain alkyl amine controlled pore glass (LCAA-CPG) beads through a stable phosphoramidate diester link. The resulting solid support was found suitable for the automated synthesis of 3′-phosphate unmodified as well as base-sensitive oligonucleotides. Efficient cleavage from the solid support was achieved by photolysis.

Sugar modified oligonucleotides. III (1). Synthesis, nuclease resistance and base pairing properties of α- and β-L-octathymidylates

Abstract Two unatural L-oligodeoxyribonucleotides, namely α- and β-L-dT8pO(CH2)3OH, have been synthesized. These oligomers are resistant towards nuclease degradation. They do not show any UV detectable base pairing with β-D-dA8 and poly rA.

α-anomeric DNA: β-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase

Abstract Nuclease-resistant α-anomeric DNA: β-rna hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H. RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A)12: d[G-G-(T)12-G-G], or by acid-solubility techniques, using a long substrate, poly(A): poly(dT). Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange. Our results suggest that RNase H, for which DNA: RNA duplexes are the natural substrates, binds to non-physiological α-DNA: RNA hybrids and is consequently inhibited. These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities. However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded α-DNA itself. Unexpectedly, polymerase activity is highly stimulated by α-oligos, analogous in their sequence to the β primer used at a concentration unable to generate a detectable synthesis. These results suggest that the inhibition of reverse transcriptase activity with the α: β may occur at different levels.

Preparation of a short synthetic apurinic oligonucleotide

The synthesis of the model apurinic oligonucleotide Tp(AP)pT is reported. Furthermore during the course of purification of this compound we have shown that the adduct formed upon reaction with methoxyamine has a Schiff base structure.