Enzymatic amplification of translation inhibition of rabbit beta-globin mRNA mediated by anti-messenger oligodeoxynucleotides covalently linked to intercalating agents.

The effects of anti-messenger oligodeoxynucleotides, covalently linked to an intercalating agent, on translation of rabbit beta-globin mRNA, were investigated both in wheat germ extract and in microinjected Xenopus oocytes. A specific inhibition of beta-globin synthesis was observed in both expression systems with a modified 11-mer covalently linked to an acridine derivative. In injected oocytes a more efficient block was observed with this modified oligonucleotide than with its unsubstituted homolog. This was ascribed to stacking interactions of the intercalating agent with base pairs which provide an additional stabilization of the [mRNA/DNA] hybrid. We demonstrated that in wheat germ extract, the modified and unmodified oligonucleotides behaved similarly due to the presence of a high RNaseH activity. RNaseH was also present, although to a lesser extent, in the oocyte cytoplasm. This anti-messenger DNA-induced degradation of target mRNA resulted in amplified efficiency of hybrid-arrested translation. This additional mechanism might provide anti-sense DNAs with an advantage over anti-sense RNAs.     

Anti-messenger oligodeoxynucleotides: specific inhibition of rabbit beta-globin synthesis in wheat germ extracts and Xenopus oocytes

Oligodeoxyribonucleotides complementary to the initiation region of rabbit beta-globin messenger RNA were used to selectively inhibit translation in a wheat germ extract and in injected Xenopus oocytes. The oligonucleotides interacted specifically with their RNA target as shown by thermal denaturation studies of hybrids on nitrocellulose filters. The longest oligonucleotide used (17-mer) efficiently blocked translation both in vitro and in vivo. In contrast the shortest one (8-mer) exhibited only a limited effect. The translation block was specific. The synthesis of endogenous proteins in oocytes and that of alpha-globin in the in vitro system were not affected by anti-beta-globin oligonucleotides. A non-complementary oligonucleotide had no inhibitory effect.     

RNase H is responsible for the non-specific inhibition of in vitro translation by 2'-O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers.

Ribonuclease H (RNase H) which recognizes and cleaves the RNA strand of mismatched RNA-DNA heteroduplexes can induce non-specific effects of antisense oligonucleotides. In a previous paper [Larrouy et al. (1992), Gene, 121, 189-194], we demonstrated that ODN1, a phosphodiester 15mer targeted to the AUG initiation region of alpha-globin mRNA, inhibited non-specifically beta-globin synthesis in wheat germ extract due to RNase H-mediated cleavage of beta-globin mRNA. Specificity was restored by using MP-ODN2, a methylphosphonate-phosphodiester sandwich analogue of ODN1, which limited RNase H activity on non-perfect hybrids. We report here that 2'-O-alkyl RNA-phosphodiester DNA sandwich analogues of ODN1, with the same phosphodiester window as MP-ODN2, are non-specific inhibitors of globin synthesis in wheat germ extract, whatever the substituent (methyl, allyl or butyl) on the 2'-OH. These sandwich oligomers induced the cleavage of non-target beta-globin RNA sites, similarly to the unmodified parent oligomer ODN1. This is likely due to the increased affinity of 2'-O-alkyl-ODN2 chimeric oligomers for both fully and partly complementary RNA, compared to MP-ODN2. In contrast, the fully modified 2'-O-methyl analogue of ODN1 was a very effective and highly specific antisense sequence. This was ascribed to its inability (i) to induce RNA cleavage by RNase H and (ii) to physically prevent the elongation of the polypeptide chain.     

Phosphoroselenoate oligodeoxynucleotides: synthesis, physico-chemical characterization, anti-sense inhibitory properties and anti-HIV activity.

Oligodeoxynucleotides with a phosphorus atom in which one of the non-bridging oxygen atoms is substituted by selenium were prepared and investigated with respect to their antisense properties. A general synthesis of phosphoroselenoate analogs of oligonucleotides is described using potassium selenocyanate as the selenium donor. The compounds, characterized by 31P NMR, were shown to decompose to phosphate with a half-life of ca. 30 days. Melting temperatures of duplexes between poly(rA) or poly(rI) with oligo(dT) and oligo(dC), respectively, indicate diminished hybridization capability of phosphoroselenoate oligomers relative to both the unmodified phosphodiester oligomers and the phosphorothioate congeners. A phosphoroselenoate 17-mer is a sequence specific inhibitor of rabbit beta-globin synthesis in wheat germ extract and in injected Xenopus oocytes. In contrast phosphoroselenoate analogs are potent non-sequence specific inhibitors in rabbit reticulocyte lysate. In vitro HIV assays were carried out on a phosphoroselenoate sequence and compared with a phosphorothioate analogue that has previously been shown to exhibit anti-HIV activity (Matsukura et al., Proc. Natl. Acad. Sci. (1987) 84, 7706-7710). The phosphoroselenoate was somewhat less active, and was much more toxic to the cells.     

RNase H-mediated inhibition of translation by antisense oligodeoxyribonucleotides: use of backbone modification to improve specificity.

A sequence of the rabbit alpha-globin mRNA is the primary target for ODN1, an unmodified 15-nucleotide (nt) antisense oligodeoxyribonucleotide (oligo). ODN1 prevented in vitro translation of both alpha- and beta-globin mRNAs in wheat germ extract. Nine secondary sites exhibiting more than 60% complementarity with ODN1 were present in the beta-globin message. The ODN1 inhibition of beta-globin synthesis was shown to be mediated by RNase H cleavage of the beta-globin mRNA at three partially complementary sites. Sandwich-type oligos consisting of a stretch of unmodified nt with a few methylphosphonate residues at both 5' and 3' ends were derived from ODN1. We have demonstrated that one such analogue (ODN2), with five phosphodiester linkages in the central region, exhibited improved specificity for alpha-globin mRNA compared with the unmodified parent 15-mer, due to a reduced ability of RNase H to cleave beta-mRNA/ODN2 mismatched duplexes.     

Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism.

We have used alpha-oligomers as antisense oligonucleotides complementary to three different sequences of the rabbit beta-globin mRNA: a region adjacent to the cap site, a region spanning the AUG initiation codon or a sequence in the coding region. These alpha-oligonucleotides were synthesized either with a free 5' OH group or linked to an acridine derivative. The effect of these oligonucleotides on mRNA translation was investigated in cell-free extracts and in Xenopus oocytes. In rabbit reticulocyte lysate and in wheat germ extracts oligomers targeted to the cap site and the initiation codon reduced beta-globin synthesis in a dose-dependent manner, whereas the target mRNA remained intact. The anti-cap alpha-oligomer was even more efficient that its beta-counterpart in rabbit reticulocyte lysate. In contrast, only the alpha-oligomer, linked to the acridine derivative, complementary to the cap region displayed significant antisense properties in Xenopus oocytes. Therefore initiation of translation can be arrested by oligonucleotide/RNA hybrids which are not substrates for RNase-H.     

The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos.

The properties of antisense phosphorothioate and unmodified oligodeoxynucleotides have been studied in Xenopus oocytes and embryos. We find that phosphorothioates, like unmodified oligodeoxynucleotides, can degrade Vg1 mRNA in oocytes via an endogenous RNase H-like activity. In oocytes, phosphorothioate oligodeoxynucleotides are more stable than unmodified oligodeoxynucleotides and are more effective in degrading Vg1 mRNA. In embryos, neither unmodified nor phosphorothioate deoxyoligonucleotides were effective in degrading Vg1 message at sub-toxic doses.     

Evaluation of some properties of a phosphorodithioate oligodeoxyribonucleotide for antisense application.

An all phosphorodithioate oligodeoxyribonucleotide (PS2; 17-mer) complementary to the coding region of the rabbit beta-globin mRNA was compared with the normal (PO2) and phosphorothioate (POS) oligonucleotide of the same size and sequence with respect to physicochemical properties and antisense activity in cell-free systems. The melting temperature (Tm) of the PS2-cDNA duplex was reduced by 17 degrees C relative to the PO2-cDNA duplex, compared to 11 degrees C for the POS-cDNA duplex, suggesting a decreased stability of the duplex with an increasing sulfur substitution. Like the POS-derivative, the PS2 oligonucleotide is quite stable against exonucleases, but these modified oligonucleotides showed different stability towards endonucleases and also towards different sub-cellular fractions of MCF-7 cells. During in vitro protein binding studies, the PS2 oligonucleotide showed similar binding (10-20%) to that of the PO2 oligonucleotide, while the POS oligonucleotide bound 60%. In cell-free translation, the PS2 oligonucleotide produced slightly higher specific translation inhibition of rabbit beta-globin mRNA compared to that of the PO2 oligonucleotide, and this was true only at concentration below 2 mM. The POS-derivative, except at 10 mM concentration, always showed higher translation arrest of the rabbit beta-globin mRNA compared to that of the other two oligonucleotides. The present study suggests that the PS2 oligonucleotide offers very little advantage over the POS oligonucleotide for use as an antisense analog.     

Antisense oligodeoxyribonucleotide-directed cleavage of maternal mRNA in Xenopus oocytes and embryos

We have investigated the effect of specific antisense oligodeoxynucleotides (oligos) on endogenous histone H4 mRNA in Xenopus oocytes, eggs and embryos. In unfertilised eggs and non-matured oocytes, one 20-mer oligo (H4-1) mediated the RNAse H-like cleavage of up to 95% of H4 mRNA (which included polysomal mRNA), and cleavage was still obtained when the size of the oligo was reduced to a 10-mer; no cleavage was observed with 6- and 8-mers. The residual uncleaved mRNA appeared to be completely inaccessible to H4-1 since a second injection caused no further cleavage. A second 20-mer (H4-2) directed against a different region of H4 mRNA was much less effective (less than 5% cleavage). In fertilised embryos, injections of H4-1 and an oligo directed against the localised Vg1 mRNA caused less cleavage than in oocytes and also showed signs of inducing localised, non-specific mRNA cleavage. However we have been able to prepare fertilised embryos devoid of Vg1 mRNA by maturing and fertilising oligo-injected oocytes in vitro.     

Effects of oligo sequence and chemistry on the efficiency of oligodeoxyribonucleotide-mediated mRNA cleavage.

Using the endogenous histone H4 mRNA of Xenopus oocytes as a target, we have previously shown that 20mer oligos complementary to different parts of this sequence vary in their effectiveness at causing mRNA cleavage in vivo, and that some of the RNA can never be cleaved. In this paper we show that the resistant RNA is not localised within one part of the oocyte, and that the relative resistance in vivo of endogenous or synthetic H4 mRNA to the different oligos is preserved in an in vitro assay system using deproteinised RNA. If an prior annealing step is included in vitro, all resistance is abolished. Chemical modification of one oligo by end substitution with methylphosphonate or phosphorothioate residues did not improve cleavage efficiency. Oligos with complete phosphorothioate substitution cause slower cleavage in vivo but persist for longer. Consequently phosphorothioate oligos are effective at lower doses than phosphodiester ones, provided that the incubation time is long enough (24 hours). Increasing oligo length from 20nt to 30nt increases phosphorothioate oligo efficiency over long reaction times in vivo, but decreases efficiency during short in vitro assays. Similar increases in length did not affect phosphodiester oligo performance in vivo, but caused a decrease in efficiency in vitro which was overcome by an annealing step.     

Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5'' noncoding region.

Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction.     

NMR investigations of duplex stability of phosphorothioate and phosphorodithioate DNA analogues modified in both strands.

Duplex formation from the self-complementary 12mer d(CGCGAATTCGCG) (Dickerson dodecamer) in which all phosphodiester linkages were replaced by phosphorothioate or phosphorodithioate linkages was studied using variable-temperature 1H and 31P NMR spectroscopy. Melting temperatures of the dodecamer, measured spectrophotometrically, showed significant decrease upon sulfur substitution (Tm 49 degrees C for the phosphorothioate and 21 degrees C for the phosphorodithioate, compared with 68 degrees C for the unmodified oligomer, in 1 M salt). Hyperchromicity observed upon melting of the dithioate was surprisingly low. NOESY spectra of the monothioate showed a cross-peak pattern characteristic for a right-handed duplex. Imino proton resonances of the duplex, shown by the mono- and the dithioate, were similar to those of the parent compound. In spite of monophasic melting curves, temperature dependence of the imino proton resonances and phosphorus resonances of the phosphorodithioate indicated heterogeneity with respect to base-pairing, compatible with the presence of a hairpin loop. Relaxation times (T1) of the imino protons in the phosphorothioate, determined by the saturation recovery method, were considerably shorter than in the unmodified oligomer. Base-pair lifetimes in the unmodified Dickerson dodecamer, determined by catalyst-dependent changes in relaxation rates of imino protons, were in the range of 2-30 ms at 20 degrees C. Strongly reduced base-pair lifetimes were found in the phosphorothioate analogue.     

Polyethylenimine but not cationic lipid improves antisense activity of 3'-capped phosphodiester oligonucleotides

Lipofectin, which is a mixture of neutral lipid with a cationic lipid, has been widely used to enhance cellular delivery of phosphorothioate, 2'-sugar-modified, and chimeric antisense oligonucleotides. Phosphodiester oligonucleotides delivered with Lipofectin usually do not elicit antisense activity probably because cationic lipid formulations do not sufficiently protect unmodified oligonucleotides from nuclease degradation. We show that a cationic polymer, polyethylenimine (PEI), improves the uptake and antisense activity of 3'-capped 20-mer and 12-mer antisense phosphodiester oligonucleotides (PO-ODN) targeted to different regions of Ha-ras mRNA and to the 3'-untranslated region (3'-UTR) of C-raf kinase. In contrast, PEI, which forms a very stable complex with the 20-mer phosphorothioate oligonucleotide (PS-ODN), does not enhance its antisense activity. Using fluorescently labeled carriers and ODN, we show that PEI-PS-ODN particles are very efficiently taken up by cells but PS-ODN is not dissociated from the carrier. Our results indicate that carrier-ODN particle size and stability and ODN release kinetics vary with the chemical nature of the ODN and the carrier being transfected into the cells. The very low cost of PEI compared with cytofectins and the increased affinity for target mRNA and decreased affinity for proteins of PO-ODN compared with PS-ODN make the use of PEI-PO-ODN very attractive.     

Selective binding of trisamine-modified phosphorothioate antisense DNA to target mRNA improves antisense activity and reduces toxicity

Antisense activity in living cells has been thought to occur via a mechanism involving both DNA-mediated hybridization arrest of target mRNA and RNase H-mediated mRNA digestion. Therefore an ideal antisense agent should be permeable to the cell and possess capacities (1) to form a thermally stable duplex in vivo with its target, (2) to discriminate between mRNAs with different degrees of complementarity, and (3) to form antisense/RNA complexes that are susceptible to RNase H hydrolysis. A trisamine-modified deoxyuridine derivative of a novel phosphorothioate DNA 15-mer that meets all these criteria is described here. Compared with the unmodified phosphorothioate oligomer, the phosphorothioate derivative exhibits a higher antisense activity as well as reduced cytotoxicity in cells infected with HIV-1. Our data suggest that the melting temperature (T(m)) between antisense DNA and the target mRNA is not only one of the factors contributing to this derivative's improved antisense activity. Also important are an enhanced ability to discriminate between sequences and an increased susceptibility of the DNA/mRNA complex to RNase H hydrolysis. These results will be useful in designing more active, clinically useful antisense drugs.     

Effects of ribosomal wash factors and spermidine on endogenous and exogenous mRNA stimulated protein synthesis in the wheat germ cell-free system.

Differential effects of Mg2+, spermidine, and reticulocyte ribosomal wash factors on the translation of endogenous, myeloma, and globin mRNA's have been observed in studies with the wheat germ cell-free protein synthesizing system. Spermidine stimulated globin mRNA translation but not the translation of endogenous wheat germ messages, and the polyamine actually inhibited the translation of myeloma mRNA. Ribosomal wash factors, on the other hand, stimulated endogenous and myeloma mRNA dependent protein synthesis in an Mg2+-dependent fashion but inhibited globin mRNA translation. The combination of ribosomal wash factors and spermidine was either stimulatory or inhibitory depending on the Mg2+ concentration and the message. It was further observed that translation of exogenous myeloma mRNA proceeded for only 60 min at 25 degrees C under all conditions tested in this study, while translation of endogenous wheat germ messages continued for longer periods of time. No differential effects of spermidine on the synthesis of high molecular weight myeloma proteins were observed.