Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro.

We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.     

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.     

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.     

Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences

Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.     

Fast and accurate determination of sites along the FUT2 in vitro transcript that are accessible to antisense oligonucleotides by application of secondary structure predictions and RNase H in combination with MALDI-TOF mass spectrometry

Alteration of gene expression by use of antisense oligonucleotides has considerable potential for therapeutic purposes and scientific studies. Although applied for almost 25 years, this technique is still associated with difficulties in finding antisense-effective regions along the target mRNA. This is mainly due to strong secondary structures preventing binding of antisense oligonucleotides and RNase H, playing a major role in antisense-mediated degradation of the mRNA. These difficulties make empirical testing of a large number of sequences complementary to various sites in the target mRNA a very lengthy and troublesome procedure. To overcome this problem, more recent strategies to find efficient antisense sites are based on secondary structure prediction and RNase H-dependent mechanisms. We were the first who directly combined these two strategies; antisense oligonucleotides complementary to predicted unpaired target mRNA regions were designed and hybridized to the corresponding RNAs. Incubation with RNase H led to cleavage of the RNA at the respective hybridization sites. Analysis of the RNA fragments by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, which has not been used in this context before, allowed exact determination of the cleavage site. Thus the technique described here is very promising when searching for effective antisense sites.     

Mechanism for suppression mRNA translation with antisense oligonucleotides

Experimental studies of the effects of antisense oligonucleotides on translation of mRNAs in cell-free systems are reviewed. Oligonucleotides complementary to the leader sequences or to the sequence overlapping the initiating codon region of mRNAs inhibit translation of the messengers. In the presence of ribonuclease H, oligodeoxyribonucleotides and their phosphorothioate analogs complementary either to the mentioned mRNA regions or to the mRNA coding sequence suppress the translation due to the RNAs cleavage. This inhibition-enhancing mechanism does not operate in the case of the oligonucleotide analogs--oligonucleoside methylphosphonates and oligonucleotides built of the alpha-nucleosides, since the complexes formed by RNA and these analogs are not substrates of the ribonuclease H. The translation inhibition efficiency is determined by the oligonucleotides lengths and by the availability of the complementary sequence in the mRNA structure. The oligonucleotides inhibitory power can be improved by the coupling to the oligonucleotides of the intercalating groups and the reactive groups.     

Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

Rational design of point mutation-selective antisense DNA targeted to codon 12 of Ha-ras mRNA in human cells.

Antisense oligodeoxynucleotides targeted to Ha-ras mRNA have been designed to discriminate between the codon 12-mutated oncogene and the normal proto-oncogene. An in vitro assay using two different sources of RNase H (rabbit reticulocyte lysates and nuclear extract from HeLa cells) was used to characterize oligonucleotide binding to normal and mutated Ha-ras mRNA. Short oligonucleotides (12- or 13mers) centered on the mutation had a very high discriminatory efficiency. Longer oligonucleotides (16mers) did not discriminate efficiently between the mutated and the normal mRNA. We have tested the efficacy of dodecanucleotides to induce RNase H cleavage of the full-length mRNA, moving the target sequence from the loop to the stem region which is formed in the vicinity of mutated codon 12. The most selective oligonucleotides were centered on the mutation which is located near the junction between the loop and stem regions even though they were less efficient at inducing RNase H cleavage than those targeted to the loop region. The 12mer antisense oligonucleotide with the highest discriminatory power was selected for cell culture studies. This oligonucleotide inhibited the proliferation of a human cell line which had been transformed with the mutated Ha-ras gene (HBL100ras1) but had no effect on the parental cell line which was transfected with the vector DNA (HBL 100neo) and expressed only the normal Ha-ras gene. Growth inhibition of HBL100ras1 cells was associated with specific ablation of targeted Ha-ras mRNA as shown by RT-PCR. These results show that 'in vitro' evaluation using an RNase H assay allowed us to select an antisense oligonucleotide which elicited a selectivity towards point-mutated Ha-ras mRNA when added at 10 microM concentration to the culture medium of cells expressing wild type and mutated Ha-ras mRNA.     

Targeted degradation of mRNA in Xenopus oocytes and embryos directed by modified oligonucleotides: studies of An2 and cyclin in embryogenesis.

We have designed antisense oligodeoxyribonucleotides which are both highly resistant to nucleolytic degradation and also serve as substrates for ribonuclease H. Using these compounds we have targeted the specific degradation of several maternal mRNAs present in Xenopus laevis oocytes and early embryos. Several internucleoside linkages at both the 3' and 5' ends of the oligonucleotides were modified as phosphoramidates to provide complete protection against exonucleases, the predominant nucleolytic activity found in both oocytes and embryos. Eight Internal linkages were left unmodified to provide a substrate for RNase H. Degradation of specific embryonic mRNAs was accomplished using subtoxic amounts of the modified oligonucleotides. Specific depletion of An2, a localized mRNA encoding the alpha subunit of the mitochondrial ATPase, produced embryos that gastrulated later than control embryos and arrested in development prior to neurulation. A modified oligonucleotide targeting Xenopus cyclin B1 and cyclin B2 mRNA was also synthesized. Following the injection of one blastomere of a two-cell embryo with the anti-cyclin oligonucleotide, cell division in that half of the embryo was inhibited, demonstrating the in vivo importance of these cyclins in mitosis. The oligonucleotide analogs described here should be useful in studying developmentally significant proteins in Xenopus.     

Taking control of gene expression with light-activated oligonucleotides

The recent development of caged oligonucletides that are efficiently activated by ultraviolet (UV) light creates opportunities for regulating gene expression with very high spatial and temporal resolution. By selectively modulating gene activity, these photochemical tools will facilitate efforts to elucidate gene function and may eventually serve therapeutic aims. We demonstrate how the incorporation of a photocleavable blocking group within a DNA duplex can transiently arrest DNA polymerase activity. Indeed, caged oligonucleotides make it possible to control many different protein-oligonucleotide interactions. In related experiments, hybridization of a reverse complementary (antisense) oligodeoxynucleotide to target mRNA can inhibit translation by recruiting endogenous RNases or sterically blocking the ribosome. Our laboratory recently synthesized caged antisense oligonucleotides composed of phosphorothioated DNA or peptide nucleic acid (PNA). The antisense oligonucleotide, which was attached to a complementary blocking oligonucleotide strand by a photocleavable linker, was blocked from binding target mRNA. This provided a useful method for photomodulating hybridization of the antisense strand to target mRNA. Caged DNA and PNA oligonucleotides have proven effective at photoregulating gene expression in cells and zebrafish embryos.     

Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity

Previous experiments have shown that mRNA translation in frog oocytes can be inhibited by the injection of a complementary antisense RNA. Here we explore the use of antisense RNAs to study the functions of localized maternal mRNAs during postfertilization development. While developmental abnormalities were observed in injected fertilized eggs, these abnormalities could not be attributed to the antisense RNA since they were induced at a similar frequency in control embryos. Biochemical tests show that the injected antisense RNA does not form stable hybrids in vivo with its complementary endogenous mRNA. In addition, a novel activity that unwinds RNA:RNA duplexes was found. This activity exists at high levels in eggs and early embryos and is absent or very much diminished in oocytes and late blastula embryos. These results suggest that antisense RNAs may be of limited use in studying the functions of maternal RNAs in Xenopus.     

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.     

Thermodynamic and Kinetic Characterization of Antisense Oligodeoxynucleotide Binding to a Structured mRNA

Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit β-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed.     

Oligonucleotide-targeted degradation of U1 and U2 snRNAs reveals differential interactions of simian virus 40 pre-mRNAs with snRNPs.

We have investigated the roles of U1 and U2 snRNP particles in SV40 pre-mRNA splicing by oligonucleotide-targeted degradation of U1 or U2 snRNAs in Xenopus laevis oocytes. Microinjection of oligonucleotides complementary to regions of U1 or U2 RNAs either in the presence or absence of SV40 DNA resulted in specific cleavage of the corresponding snRNA. Unexpectedly, degradation of U1 or U2 snRNA was far more extensive when the oligonucleotide was injected without, or prior to, introduction of viral DNA. In either co-injected or pre-injected oocytes, these oligonucleotides caused a dramatic reduction in the accumulation of spliced SV40 mRNA expressed from the viral late region, and a commensurate increase in unspliced late RNA. When pre-injected, two different U2 specific oligonucleotides also inhibited the formation of both large and small tumor antigen spliced early mRNAs. However, even when, by pre-injection of a U1 5' end-specific oligonucleotide, greater than 95% degradation of the U1 snRNA 5' ends occurred in oocytes, no reduction in early pre-mRNA splicing was observed. In contrast, the same U1 5' end oligonucleotide, when added to HeLa splicing extracts, substantially inhibited the splicing of SV40 early pre-mRNA, indicating that U1 mRNP is not totally dispensable for early splicing. These findings confirm and extend our earlier observations which suggested that different pre-mRNAs vary in their requirements for snRNPs.     

Translation of cyclin mRNA is necessary for extracts of activated xenopus eggs to enter mitosis

The cyclins are a family of proteins encoded by maternal mRNA. Cyclin polypeptides accumulate during interphase and are destroyed during mitosis at about the time of entry into anaphase. We show here that Xenopus oocytes contain mRNAs encoding two cyclins that are major translation products in a cell-free extract from activated eggs. Cutting these mRNAs with antisense oligonucleotides and endogenous RNAase H blocks entry into mitosis in a cell-free egg extract. The extracts can enter mitosis if either of the cyclin mRNAs is left intact. We conclude that the synthesis of these cyclins is necessary for mitotic cell cycles in cleaving Xenopus embryos.