alpha-Oligodeoxynucleotides.
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Biochemistry↗
Biomedical subjects
B Rayner
Publications and source records attributed to B Rayner.
Mutagenicity of a unique apurinic/apyrimidinic site in mammalian cells.
Abasic sites are common DNA lesions produced either spontaneously or as a consequence of the action of some genotoxic agent. The mutagenic properties of a unique abasic site replicated in mammalian cells have been studied using a shuttle vector. A plasmid, able to replicate both in mammalian cells and in bacteria, carrying a unique abasic site chemically synthesized has been constructed. After replication in mammalian cells, plasmid DNA was recovered and used to transform bacteria. Mutants were screened without selection pressure by differential hybridization with a labelled oligonucleotide and their DNA was sequenced. A mutation frequency ranging from 1% to 3% was found, depending on the base originally inserted during the vector construction, opposite the abasic site. All the sequenced mutants correspond to single base-pair substitutions targeted at the abasic site. We observed a deficit in guanine incorporation opposite the abasic site, while the three other bases were incorporated with a similar efficiency. The mutational potency of abasic sites was observed without any voluntary preconditioning treatment of mammalian cells in order to induce "SOS" like conditions.
Animals↗
Synthesis and base-pairing properties of the nuclease-resistant alpha-anomeric dodecaribonucleotide alpha-[r(UCUUAACCCACA)].
The non natural oligoribonucleotide alpha-[r(UCUUAACCCACA)] consisting exclusively of alpha-anomeric ribonucleoside units was synthesized according to the phosphoramidite methodology and the solid support technology. For this purpose, the base-protected alpha-ribonucleosides were synthesized and converted into their O-methylphosphoramidites. Assembling was carried out on a DNA synthesizer with an average efficiency of 97% per step. Base composition of this nuclease-resistant alpha-RNA strand was ascertained after chemical and enzymatic hydrolysis and HPLC analysis of the hydrolysate. Whereas no spectroscopic evidence of base pairing was found above 0 degrees C between alpha-[r(UCUUAACCCACA)] and beta-[d(TGTGGGTTAAGA)], a clear UV absorbance transition (Tm 25.5 degrees C) was observed during the hybridization of the same alpha-RNA strand with beta-[d(AGAATTGGGTGT)]. In this latter case, the mixing curve titration suggests formation at low temperature of a triplex involving two alpha-RNA and one beta-DNA strands. Moreover, this alpha-decaribonucleotide complementary in parallel orientation of the splice receptor of HIV-1 tat mRNA was found to inhibit (10 microM less than ED50 less than 20 microM), with apparent lack of sequence specificity, the de novo HIV-1 infection in cultured cells.
Base Composition↗
Structure and conformation in solution of the parallel-stranded hybrid alpha-d(CGCAATTCGC).beta-d(GCGTTAAGCG) by high-resolution 2D NMR.
The unnatural duplex oligonucleotide alpha-d(CGCAATTCGC).beta-d(GCGTTAAGCG) was analyzed by high-resolution NMR methods. All of the exchangeable imino and nonexchangeable protons of the duplex were assigned. Detection of all 10 of the exchangeable imino protons confirms that a parallel, unsymmetrical duplex is formed. The thermal stability of the parallel duplex is similar to the analogous antiparallel beta-beta duplex. The right handedness of the helix is confirmed by inter-residue [H8/H6-H1'] and [H8/H6-H2"] NOEs to the 5'-neighbor in the beta-strand and [H8/H6-H1'] NOEs to the 3'-neighbor in the alpha-strand. Intra-residue and inter-residue distances between base protons and deoxyribose protons in both strands were determined using the isolated spin-pair approximation for NOESY cross peaks acquired with mixing times 50 ms or less. The NOE data are consistent with a B-form geometry adopted by the alpha/beta hybrid decamer.
Base Sequence↗
9-Aminoellipticine-derivatized alpha- and beta-oligodeoxyribonucleotides targeted to the cap of beta-globin mRNA: hybridization to natural and engineered mRNA, inhibition of translation, and improved effect of tandem chains.
We studied the duplex stability and the antimessenger activity of 9-aminoellipticine-5'-functionalized alpha- and beta-anomeric DNA sequences complementary to the first 14 nucleotides of the rabbit beta-globin mRNA. The duplex formed by the beta-conjugate with the natural mRNA target possessed a marginally better stability to that of the duplex formed by the unfunctionalized compound, as measured by the thermal elution. The alpha-conjugate did not anneal to native mRNA, possibly due to the interference of the 9-aminoellipticine with the cap structure and, unlike the beta-adduct, was practically inactive as inhibitor of translation in a cell-free system. However, it did hybridize to an RNA construction containing the beta-globin mRNA plus an additional 50 bases in 5'. Surprisingly, translation from this construction was inhibited by the alpha-species in spite of the nonvicinity of the target to the cap. Both alpha and beta conjugates hybridized to a DNA 14-mer of the same sequence as that targeted onto the mRNA. Thermal denaturation and fluorescence spectroscopy showed that the drug brought no considerable stabilization to the duplex, the linker presumably being unfavorable to intercalation. An increased stability of the complex and a higher inhibitory effect on cell-free beta-globin translation were obtained with two contiguous beta-oligomers of which one was functionalized.
Animals↗
Inhibition of murine leukemia viruses by nuclease-resistant alpha-oligonucleotides.
We studied the antiviral activity of nuclease-resistant alpha-anomeric oligonucleotides. An alpha-oligonucleotide (20-mer) targeted to the primer binding site (PBS) of murine retroviruses inhibited viral spreading. The inhibition only occurred when the cells had been electropermeabilized in the presence of the oligonucleotide. The PBS sequence is involved in reverse transcription and in translation. The data suggest that the oligonucleotide could perturb reverse transcription activity. Thus, either the oligonucleotide induced a decrease in initiation or it inhibited the extension of the minus or plus strands DNA during reverse transcription. These results show that reverse transcription may be an interesting target for antisense oligonucleotides.
3T3 Cells↗
Comparison of anti-RNA properties of normal and ellipticine functionalized alpha and beta-oligonucleotides.
RNA is not cleaved as a consequence of the binding of RNase H to the duplex between RNA and a complementary alpha-oligodeoxyribonucleotide (oligo). In consequence targets have been selected which do not a priori require the action of RNase H to inhibit genetic expression. Two models have been used: The Friend Murine Leukemia Virus (F-MuLV) and the synthesis of rabbit beta globin.alpha-oligos trigger specific inhibitions in both systems. The functionalisation in 5' with the intercalating agent 9-NH2-ellipticine renders the oligos resistant to degradation and allows a direct action on cells.
Animals↗
Alpha-oligonucleotides: a unique class of modified chimeric nucleic acids.
alpha-Anomeric oligonucleotides are resistant to nucleases and display parallel hybridization with complementary DNA and RNA sequences. Although alpha-DNA/beta-RNA duplexes are not substrates for RNases H, alpha-oligos are able to inhibit translation through a RNase H independent mechanism. alpha-Oligos and their alpha-phosphorothioate analogs (12 mer) targeted against the splice acceptor site of the HIV-TAT gene were potent inhibitors of de novo HIV infection. Furthermore alpha-phosphorothioate and dithioate homo-oligomers exhibit an in vitro, nonsequence-specific, inhibitory effect on HIV reverse transcriptase.
Base Sequence↗
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.
Animals↗
Polarity of annealing and structural analysis of the RNase H resistant alpha-5'-d[TACACA].beta-5'-r[AUGUGU] hybrid determined by high-field 1H, 13C, and 31P NMR analysis.
The novel hybrid duplex alpha-5'-d[TACACA]-3'.beta-5'-r[AUGUGU]-3' was analyzed extensively by 1D and 2D NMR methods. Two forms of the duplex exist in about an 80:20 ratio. Analysis of the exchangeable imino protons of the major component revealed that three AU and one AT base pair are present in addition to two GC base pairs, confirming that the duplex anneals in parallel orientation. The presence of the AT base pair, which can only be accounted for by a parallel duplex, was confirmed by a selective INEPT experiment, which correlated the thymidine imino proton to its C5 carbon. The lesser antiparallel form could be detected by exchangeable and nonexchangeable proton resonances in both strands. An exchange peak was observed in the NOESY spectrum for the thymidine methyl group resonance in both the predominant and lesser conformations, indicating the lifetime of the individual structures was on the millisecond time scale. The nonexchangeable protons of the predominant duplex were assigned by standard methods. The sugar pucker of the ribonucleosides was determined to be of the "S" type by a pseudorotation analysis according to Altona, with the J-couplings measured from the multiplet components of the phase-sensitive COSY experiment. The NOE pattern observed for the alpha-deoxynucleosides also suggested an S-type sugar pucker. The adoption of an S-type sugar pucker for both strands indicates that, in contrast to RNA.DNA duplexes formed exclusively from beta-nucleotides, the alpha-DNA.beta-RNA duplex may form a B-type helix. The 31P resonances of the alpha and beta strands have very different chemical shifts in the hybrid duplex and the difference persists above the helix melting temperature, indicating an intrinsic difference in 31P chemical shift for nucleotides differing only in the configuration about the glycosidic bond.
Base Composition↗
Sugar modified oligonucleotides. III (1). Synthesis, nuclease resistance and base pairing properties of alpha- and beta-L-octathymidylates.
Two unnatural L-oligodeoxyribonucleotides, namely alpha- and beta-L-dT8pO(CH2)3OH, have been synthesized. These oligomers are resistant towards nuclease degradation. They do not show any UV detectable base pairing with beta-D-dA8 and poly rA.
Animals↗
Alpha-oligodeoxynucleotides (alpha-DNA): a new chimeric nucleic acid analog.
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Animals↗
Synthesis, thermal stability and reactivity towards 9-aminoellipticine of double-stranded oligonucleotides containing a true abasic site.
A 13 mers abasic oligonucleotide was synthetized. It was therefore possible to compare thermal stability and reactivity of duplex oligonucleotides either with an apurinic/apyrimidinic site or without any lesion. An important decrease in the melting temperature appeared for duplexes with an abasic site. The chemical reaction of these modified oligonucleotides with the intercalating agent 9-aminoellipticine was studied by gel electrophoresis and by fluorescence. The formation of a Schiff base between 9-aminoellipticine and abasic sites was rapid and complete with duplexes at 11 degrees C. Schiff base related fluorescence and beta-elimination cleavage were more important with the apyrimidinic sites than with the apurinic ones. When compared to previous results obtained with the model d(TprpT) some unexpected behaviours appeared with longer and duplex oligonucleotides. For instance only partial beta-elimination cleavage was observed. It is likely that stacking parameters in the double helix play a great role in the studied reaction.
Alkaloids↗
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.
Base Composition↗
Comparative activity of alpha- and beta-anomeric oligonucleotides on rabbit beta globin synthesis: inhibitory effect of cap targeted alpha-oligonucleotides.
Alpha-anomeric oligonucleotides are resistant to nucleases and display parallel annealing to RNA complementary sequences. We compared the effect of alpha- and beta-oligonucleotides targeted against various mRNA regions on the rabbit beta globin in vitro synthesis. In order to determine the role of RNase H, experiments were performed in both rabbit reticulocyte lysate and wheat germ extract. As expected beta-oligonucleotides were found more efficient in wheat germ extract which is rich in RNase H activity and alpha-oligonucleotide targeted against the initiation codon or downstream had no effect because they do not induce mRNA cleavage by RNase H. However, we report, for the first time, a specific translation inhibition by alpha-oligonucleotides. This occurs provided they are targeted against the cap region in 5' of the mRNA.
Animals↗
alpha-DNA X: alpha and beta tetrathymidilates covalently linked to oxazolopyridocarbazolium (OPC): comparative stabilization of oligo beta-[dT]:oligo beta-[dA] and oligo alpha-[dT]:oligo beta-[dA] duplexes by the intercalating agent.
The influence of the intercalating oxazolopyridocarbazolium (HOPC) on the stabilization of modified oligonucleotides: alpha-T4c5OPC or beta-T4c5OPC associated to beta-oligo (dA) was studied. It appears that the situation is different from what has been observed for the interaction of these modified oligonucleotides with poly (rA). The higher free energy of formation of the alpha-T4c5OPC :beta-oligo(dA), when compared to beta-T4c5OPC, is essentially due to the overall stability added to this system by the intercalator. This enhanced stability comes from a higher number of binding sites of HOPC for the alpha:beta duplex together with a lower van't Hoff energy of formation of the alpha:beta duplex.
Carbazoles↗
Mechanism of cleavage of apurinic sites by 9-aminoellipticine.
We have studied the kinetics of breakage of apurinic (AP) sites by the intercalating agent 9-aminoellipticine using fluorimetric methods with single (ss)- and double (ds)-stranded apurinic DNA. In order to understand the chemical process, high performance liquid chromatography was used to follow the reaction kinetics with the apurinic oligonucleotide model T(AP)T. The unstable intermediate, which is responsible for the beta-elimination step, is a Schiff base resulting from the interaction of the amino group of the aromatic amine with the aldehyde function of the deoxyribose moiety (AP site). Fluorescence occurs simultaneously with the breakage of both ss and ds DNA and of the oligonucleotide and arises from the formation of a conjugated double bond on the Schiff base through the beta-elimination reaction. In optimal conditions, the second order rate constant for the fluorescence build up is 15 x 10(3) min-1 M-1 for ds DNA and 0.105 x 10(3) min-1 M-1 for T(AP)T. The ability of 9-aminoellipticine to induce fluorescence and breakage of ss DNA and T(AP)T shows that intercalation is not essential for this reaction to occur. Nevertheless, the greater rate constant with DNA suggests that stacking is an important parameter for the reaction of the aromatic amine with the AP site.
Alkaloids↗
Alpha-DNA.IX: Parallel annealing of alpha-anomeric oligodeoxyribonucleotides to natural mRNA is required for interference in RNase H mediated hydrolysis and reverse transcription.
ps- and aps-alpha anomeric oligodeoxyribonucleotides were designed to recognize in parallel (ps) or antiparallel (aps) orientation two different sites of a 1000 base-long mRNA. Northern blots experiments indicate that only ps-alpha-oligonucleotides were able to hybridize to the mRNA target. Furthermore, only ps-alpha-oligonucleotides were able, in a sequence specific way, to protect the mRNA target against RNase H mediated hydrolysis or to inactivate the priming capacity of beta-oligodeoxynucleotide probes in reverse transcription. Formation of parallel-stranded mRNA alpha-oligonucleotide miniduplexes which prevents hybridization of beta-oligonucleotide probes is the most likely mechanism accounting for these results. Use of alpha-oligonucleotides as potential gene control agents is discussed.
Base Sequence↗