Baculovirus directly activates murine NK cells via TLR9.

The importance of natural killer (NK) cells in innate immune responses against tumors or viral infections enhances the appeal of NK cell-based immunotherapeutic approaches. We have recently reported that baculovirus (BV)-infected dendritic cells (DCs; BV-DCs) induce antitumor immunity against established tumors in mice. These antitumor effects were CD8+ T-cell and NK cell dependent; however, they were found to be CD4+ T-cell independent. In this study, we investigated the involvement of Toll-like receptor 9 (TLR9) in the process of BV recognition by NK cells. We found that BV directly stimulated NK cells, induced the expression of the activation marker CD69 and promoted interferon-gamma (IFN-γ) production and cytotoxicity. Moreover, TLR9 knockout in mice (tlr9-/- NK cells) inhibited NK cell responses to BV, indicating that TLR9 may have a relevant role in the BV-induced upregulation of NK cell functions. Our data demonstrated for the first time that NK cells directly recognize BV via TLR9, which provides opportunities for the use of this technique as an effective tool for BV-based immunotherapies against malignancies.

Antitumor effects of baculovirus-infected dendritic cells against human pancreatic carcinoma.

Recently, we showed that baculovirus (BV)-infected dendritic cells (DCs) (BV-DCs) induced antitumor immunity against established tumors in mice. These antitumor effects were CD8(+) T-cell and natural killer (NK) cell dependent but CD4(+) T-cell independent. In the current study, we examined the antitumor effect of BV-DCs on human pancreatic cancer cells (AsPC-1). After treatment with BV-infected bone marrow-derived dendritic cells (BMDCs), human pancreatic tumors caused by AsPC-1 cells in a nude mouse model were significantly reduced in size, and the survival of the mice was improved compared with that of non-immature BMDC (iDC)- and BV-DC-immunized mice. We also found that wild-type BV could activate human DCs (HDCs) and that NK cells were activated by BV-infected HDCs (BHDCs). Our findings show that BV-DCs can induce antitumor immunity, which paves the way for the use of this technique as an effective tool for DC immunotherapy against malignancies.

Gene silencing by the tRNA maturase tRNase ZL under the direction of small-guide RNA.

We have been developing a unique system for the downregulation of a gene expression through cutting a specific mRNA by the long form of tRNA 3'-processing endoribonuclease (tRNase Z(L)) under the direction of small-guide RNA (sgRNA). However, the efficacy of this system and the involvement of tRNase Z(L) in the living cells were not clear. Here we show, by targeting the exogenous luciferase gene, that the efficacy of the sgRNA/tRNase Z(L) method can become comparable to that of the RNA interference technology and that the gene silencing is owing to tRNase Z(L) directed by sgRNA not owing to a simple antisense effect. We also show that tRNase Z(L) together with sgRNA can downregulate expression of the endogenous human genes Bcl-2 and glycogen synthase kinase-3beta by degrading their mRNAs in cell culture. Furthermore, we demonstrate that a gene expression in the livers of postnatal mice can be inhibited by an only seven-nucleotide sgRNA. These data suggest that sgRNA might be utilized as therapeutic agents to treat diseases such as cancers and AIDS.

A new class of anti-HIV-1 oligonucleotide targeted to the polypurine tract of viral RNA.

The PPT is highly conserved among the known HIV-1 strains, and is a possible target for triplex formation. We show triple-helix formation by a two-strand-system (FTFOs, DsDGloopT5-37) targeted to the polypurine tract (PPT) of HIV-1. In HIV-1 infected MOLT-4 cells, the FTFOs containing phosphorothioate groups at the antisense strand and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense phos-phorothioate oligonucleotides indicating sequence-specific inhibition of HIV-1 replication for 62 days. However, AZT, treated cells expressed high levels of p 24 products after 46 days.

Anti-HIV-1 activity of an antisense phosphorothioate oligonucleotide bearing imidazole and primary amine groups.

We have previously shown that RNA cleaving reagents with imidazole and primary amine groups on the 5'-end of antisense oligodeoxyribonucleotides could site-specifically cleave CpA as the target sequence of the substrate tRNA in vitro. In this study, a RNA cleaving reagent, composed of imidazole and primary amine groups on an antisense phosphorothioate oligonucleotide (Im-anti-s-ODN), was synthesized and evaluated for anti-HIV-1 activity in MT-4 cells. The sequence of the Im-anti-s-ODN was designed to be complementary to the HIV-1 gag-mRNA and to bind adjacent to the CpA cleavage site position. Im-anti-s-ODN encapsulated with the transfection reagent, DMRIE-C, had higher anti-HIV-1 activity than the unmodified antisense phosphorothioate oligonucleotide (anti-s-ODN) at a 2 microM concentration. Furthermore, the Im-anti-ODN encapsulated with DMRIE-C conferred sequence-specific inhibition.

Two basic regions of NCp7 are sufficient for conformational conversion of HIV-1 dimerization initiation site from kissing-loop dimer to extended-duplex dimer.

Nucleocapsid (NC) protein possesses nucleotide-annealing activities, which are used in various processes in retroviral life cycle. As conserved characters, the NC proteins have one or two zinc fingers of CX(2)CX(4)HX(4)C motif surrounded by basic amino acid sequences. Requirement of the zinc fingers for the annealing activities of NC protein remains controversial. In this study, we focused the requirement in the process of maturation of dimeric viral RNA. Discrimination between immature and mature dimers of synthetic RNA corresponding to the dimerization initiation site of human immunodeficiency virus type 1 (HIV-1) genomic RNA was performed based on their Mg(2+)-dependent stability in gel electrophoreses and on their distinct signal pattern from NMR analysis of imino protons. Chaperoning activity of the HIV-1 NC protein, NCp7, and its fragments for maturation of dimeric RNA was investigated using these experimental systems. We found that the two basic regions flanking the N-terminal zinc finger of NCp7, which are connected by two glycine residues instead of the zinc finger, were sufficient, although about 10 times the amounts of peptide were needed in comparison with intact NCp7. Further, it was found that the amount of basic residues rather than the amino acid sequence itself is important for the activity. The zinc fingers may involve the binding affinity and/or such a possible specific binding of NCp7 to dimerization initiation site dimer that leads to the maturation reaction.

Antisense therapy of influenza.

The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides (S-ODNs) with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with those directed to the PB2 target sites. The liposomally encapsulated antisense phosphorothioate oligonucleotides exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas the free antisense phosphorothioate oligonucleotides were observed to inhibit viral absorption to MDCK cells. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. Balb/c mice exposed to the influenza virus A (A/PR/8/34) strain at dose of 100 LD(50)s were treated i.v. with various doses (5-40 mg/kg) of liposomally (Tfx-10) encapsulated PB2-AUG or PA-AUG before virus infection and 1 and 3 days postinfection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in days (MDS) and increased the survival rates with a dose-dependent manner. We demonstrate the first successful in vivo antiviral activity of antisense administered i.v. in experimental respiratory tract infections induced with influenza virus A.

Mutations of p53 in morphologically non-neoplastic mucosa of long-standing ulcerative colitis.

Two cases of ulcerative colitis (UC)-associated carcinoma or dysplasia and morphologically non-neoplastic mucosa with p53 protein overexpression (MNNM-p53OE) were selected. DNA was extracted from the paraffin blocks of these lesions and exons 5 - 8 of the p53 gene were analyzed by PCR and direct sequencing. In addition, mutations in K-ras codon 12 were analyzed by PCR-RFLP methods. MNNM-p53OE was located surrounding and adjoining a coexisting carcinoma and / or dysplasia. A p53 mutation was detected in 12 / 22 (54.5%) MNNM-p53OE samples, 4 / 8 (50%) dysplasia samples and 8 / 8 (100%) carcinoma samples. The p53 mutations detected in MNNM-p53OE were identical to those demonstrated in the adjoining carcinoma and / or dysplasia. No K-ras codon 12 mutation was detected in any of the samples. These results indicate that MNNM-p53OE may share an identical clonal linkage with a coexisting carcinoma and / or dysplasia, and may be an initial and submorphological form of UC-associated neoplasia. Recognition of MNNM-p53OE in biopsy specimens may help to identify patients with UC at risk of developing colorectal carcinoma.

Heterogeneity of p53 mutational status in intramucosal carcinoma of the colorectum.

The aim of this study was to elucidate whether or not p53 genetic heterogeneity would occur while colorectal carcinoma was limited to the mucosa. Eight cases of endoscopically resected colorectal intramucosal carcinomas were analyzed to determine the p53 gene sequence (exons 5 to 8). Six out of 8 cases showed p53 gene mutations, and in all of them, the mutational status was heterogeneous. In 4 cases, mutated codons were heterogeneous as well. These data indicate that p53 gene alterations in colorectal carcinomas occur and diverge at the stage of intramucosal carcinoma, supporting our previously proposed hypothesis that colorectal carcinomas can be composed of various subclones as regards p53 gene mutation, while the carcinoma is limited to the mucosa, and one of these subclones commences invasion to the submucosa after clonal selection, thus generating a monoclonal invasive carcinoma.

Conformational change of dimerization initiation site of HIV-1 genomic RNA by NCp7 or heat treatment.

Dimerization initiation site (DIS) of the human immunodeficiency virus type 1 (HIV-1) genome has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. A DIS RNA fragment spontaneously forms a kissing dimer and is converted into an extended-duplex dimer by supplement of nucleocapsid protein NCp7. This two-step dimerization reaction can be also executed by a heat treatment instead of the binding proteins. However, it has not identified whether mechanisms of the conformational conversion by two different treatments are identical or not. In the present study, we used a series of DIS RNA oligonucleotides and the conformations of two extended-duplex dimers produced by the two different treatments were compared by the analysis of NMR spectra in the imino proton region. It was found that the effects of the two kinds of treatments are quite similar and the conformations of the two extended-duplex dimers are identical. These findings suggest that the conversion mechanisms DIS RNA by NCp7 and heat treatments are similar.

Early colorectal cancer with special reference to the superficial nonpolypoid type from a histopathologic point of view.

The incidence and histopathologic characteristics of nonpolypoid (superficial type) early colorectal carcinomas were studied and compared with those of the polypoid type. The superficial type was subclassified as elevated (type IIa), type IIa with central depression (type IIa + IIc), plain (type IIb), depressed (type IIc), and IIc with marginal elevation (type IIc + IIa). The superficial type comprised 22% and 27% of intramucosal and submucosal carcinomas, respectively. Pure type IIb was not found, and there were only three pure type IIc lesions. Type IIa + IIc and IIc + IIa (and IIc) showed a significantly higher rate of submucosal invasion among the small tumors (59% and 71% less than 20 mm, respectively) compared to the polypoid type; type IIa showed no significant difference. The incidence of lymph node metastasis among submucosal carcinomas showed no significant difference between the superficial type and the polypoid type. About 64% and 52% of type IIa and IIa + IIc tumors accompanied residual adenoma, suggesting that they originated from small, flat adenomas through the adenoma-carcinoma sequence, whereas type IIc + IIa (and IIc) did not have an adenomatous component, implying that they arose de novo or originated through an adenoma-carcinoma sequence at a smaller size than the type IIa and IIa + IIc lesions. Superficial-type early colorectal carcinomas are not rare, and they are not uniform in nature. Rapid growth and invasion to the submucosa is characteristic of superficial-type lesions with a central depression, and only superficial depressed (type IIc + IIa, IIc) lesions can arise de novo. Although they grow rapidly to invade the submucosa, it cannot be said that they show more aggressive behavior than the polypoid type.

Interactions of a didomain fragment of the Drosophila sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective [5-2H]uridine substitutions.

Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sx1) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5'-terminus of a uridine stretch. First, we prepared a [5-2H]uridine phosphoramidite, and synthesized a series of 2H-labeled RNAs, in which all of the uridine residues except one were replaced by [5-2H]uridine in the target sequence, GU8C. By observing the H5-H6 TOCSY cross peaks of the series of 2H-labeled RNAs complexed with the Sx1 RBDI-RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU2GU8, AU8, and UAU8, were assigned by comparison with those of GU8C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1' resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2'-endo ribose conformation in the complex.

Inhibition of human telomerase activity by antisense phosphorothioate oligonucleotides encapsulated with the transfection reagent, FuGENE6, in HeLa cells.

Telomerase, a ribonucleoprotein, synthesizes telomeric repeats (TTAGGG) onto the ends of chromosomes to maintain the constant length of the telomere DNA, and its activity is detectable in approximately 85%-90% of primary human cancers. Thus, it is postulated that human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphorothioate oligonucleotides (S-ODN) were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The S-ODN were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 (Boehringer Mannheim, Mannheim, Germany) was used to enhance the cellular uptake of the oligonucleotides in cell cultures. The S-ODN encapsulated with FuGENE6 clearly inhibited telomerase activity in HeLa cells and showed sequence-specific inhibition. The encapsulated S-ODN-3 with a 19-nucleotide, (nt) chain length had inhibitory effects similar to those of the 21-mer and 23-mer S-ODN sequences (S-ODN-4 and 5), but the 15-mer and 17-mer S-ODN sequences (S-ODN-1 and 2) failed to satisfactorily prevent telomerase activity. However, apoptotic HeLa cell death was not associated with telomerase inhibition. Furthermore, the encapsulated S-ODN did not appear to be cytotoxic in terms of the cell growth rate. The oligonucleotides encapsulated with the transfection reagent had enhanced cellular uptake, and cytoplasmic and nuclear localizations were observed. However, weak fluorescent signals were observed within the cytoplasms of HeLa cells treated with the free S-ODN-3. Thus, the activities of the S-ODN were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes and is appropriate for use in vitro and in vivo.

Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome.

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.

Anti-human immunodeficiency virus type 1 of a two-strand-system targeted to the polypurine tract.

The polypurine tract (PPT) is highly conserved among the known human immunodeficiency virus (HIV)-1 strains, and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand-system (FTFOs) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand-system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e., the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3'-exonuclease resistance. In the observation of the FITC-DsDGloopT5-37 with MOLT-4 cells by a confocal laser scanning microscope, diffuse fluorescence was apparently observed in the cytoplasm and nucleus. However, weak fluorescence was observed within the cytoplasm and nucleus of MOLT-4 cells treated with the antisense phosphorothioate oligonucleotides (S-ODN-gag-AUG). In HIV-1 infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.

Anti-HIV-1 activity by a triple-helix forming oligonucleotides targeted to polypurine tract on viral RNA.

Reverse transcription of HIV-1 into double-stranded DNA involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by reverse transcriptase (RT). The PPT is a possible target for triple-helix formation. We show the effects of triple-helix formation by assays of RNase H cleavage inhibition in vitro using two systems (two-strand-system (FTFOs) or three-strand-system (TFOs)) targeted to the polypurine tract (PPT) of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand-system. The FTFOs inhibited the RNase H activity in a sequence-specific manner, i.e., the triplex actually formed at the PPT and blocked the RNase H. The FTFOs containing the phosphorothioate groups at the antisense strand showed greater 3'-exonuclease resistance. In HIV-1 infected MT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense strand and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense phosphorothioate oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.

Antisense oligodeoxynucleotide complementary to CXCR4 mRNA block replication of HIV-1 in COS cells.

CXCR4 is both a chemokine receptor and an entry co-receptor for the T-cell line-adapted human immunodeficiency virus type 1 (HIV-1). To find a more efficacious therapeutic treatment of acquired immunodeficiency syndrome, we examined the effects of antisense oligonucleotides on CXCR4 production. COS cells, stably expressing CXCR4 and CD4, were incubated with several kinds of oligonucleotides. Total human p24 antigen production was determined using an enzyme-linked immunosorbent assay system. An antisense phosphorothioate-modified oligonucleotide, complementary to the translation initiation region of the CXCR4 mRNA, showed minimal inhibition of p24 antigen production at the high concentration of 2 microM. On the other hand, the antisense phosphorothioate oligonucleotide, when used with transfection reagents, showed high efficiency at low concentrations, and confirmed the sequence-specific action. Interestingly, the oligonucleotide with the natural phosphodiester backbone, when used with the transfection reagents, also had high functional effects, comparable to the modified oligonucleotide. This study defines the prerequisite criteria necessary for the design and the application of antisense oligonucleotides against HIV-1 in vivo.

Inhibition of HIV-1 replication by a two-strand system (FTFOs) targeted to the polypurine tract.

Reverse transcription of HIV-1 vRNA into the double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract (PPT) by reverse transcriptase (RT). The PPT is highly conserved among the known human immunodeficiency virus (HIV-1) strains and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand system (foldback triplex-forming oligonucleotides (FTFOs)) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e. the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3'-exonuclease resistance. In HIV-1-infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base-pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.

Specific inhibition of human telomerase activity by transfection reagent, FuGENE6-antisense phosphorothioate oligonucleotide complex in HeLa cells.

Human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphodiester (ODNs) and phosphorothioate (S-ODNs) oligonucleotides were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The ODNs and S-ODNs were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 was used to enhance the cellular uptake of oligonucleotides in cell cultures. The results showed that S-ODN-3 (19-mer) encapsulated with FuGENE6 clearly inhibited the telomerase activity in HeLa cells, and the inhibitory efficiency increased with an increase in the S-ODN-3. However, free S-ODN-3 showed no inhibitory activity. On the other hand, ODN-3 encapsulated with FuGENE6 had no detectable inhibitory activity. The encapsulated S-ODNs exhibited higher inhibitory activities than the free S-ODNs, and showed sequence specific inhibition. Thus, the activities of the S-ODNs were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, and is appropriate for use in vitro and in vivo.

Inhibition of restriction endonuclease cleavage by triple helix formation with RNA and 2'-O-methyl RNA oligonucleotides containing 8-oxo-adenosine in place of cytidine.

The ability of homopyrimidine oligoribonucleotides (RNA) and oligo-2'-O-methyl-ribonucleotides (2'-O-methyl RNA) containing 8-oxo-adenosine (AOH) and 8-oxo-2'-O-methyl (AmOH) adenosine to form stable, triple-helical structures with sequences containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The AOH- and AmOH-substituted RNA and 2'-O-methyl RNA oligonucleotides were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise in specificity. The substitutions of three cytidine residues with AOH showed higher endonuclease inhibition than the substitution of either one or two cytidine residues with AOH. In particular, the 2'-O-methyl RNA oligonucleotide with only one cytidine substituted with AmOH showed higher endonuclease inhibition than the homopyrimidine RNA and 2'-O-methyl RNA oligonucleotides and the RNA oligonucleotides containing either one or two AOH moieties. Furthermore, the AmOH-substituted 2'-O-methyl RNA oligonucleotides were stable (53%) after an incubation in 10% fetal bovine serum for 8 h, whereas the RNA oligonucleotides were completely degraded. Increased resistance to nucleases is observed with the introduction of 2'-O-methylnucleosides. This stabilization should help us to design much more efficient third strand homopyrimidine oligomer and antisense nucleic acid-based antiviral therapies, which could be used as tools in cellular biology.