LidNA, a miRNA inhibitor constructed with unmodified DNA, requires an xxxA insertion sequence in miRNA binding site for its potent inhibitory activity.

The involvement of miRNAs in the pathogenesis of various diseases, including cancer, poses the need for developing miRNA inhibitors. Previously, using unmodified DNA, we designed LidNA, which inhibited miRNA function more potently than 2'-O-methylated RNA and locked nucleic acid. LidNA consists of a complementary sequence to miRNA flanked by two structured DNAs. Alterations in the connected sequences between the complementary region and structured region modestly affect miRNA inhibition activity. Surprisingly, variations in the mismatched insertion sequence in the center of the complementary sequence significantly affect activity. The central insertion sequence xxxA is required for the potent miRNA inhibitory effects of LidNA. This suggests that both the structure and insertion sequence of LidNA and other miRNA inhibitors should be considered for maximal miRNA inhibitory activity.

Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

Influence of pendant chiral C(γ)-(alkylideneamino/guanidino) cationic side-chains of PNA backbone on hybridization with complementary DNA/RNA and cell permeability.

Intrinsically cationic and chiral C(γ)-substituted peptide nucleic acid (PNA) analogues have been synthesized in the form of γ(S)-ethyleneamino (eam)- and γ(S)-ethyleneguanidino (egd)-PNA with two carbon spacers from the backbone. The relative stabilization (ΔTm) of duplexes from modified cationic PNAs as compared to 2-aminoethylglycyl (aeg)-PNA is better with complementary DNA (PNA:DNA) than with complementary RNA (PNA:RNA). Inherently, PNA:RNA duplexes have higher stability than PNA:DNA duplexes, and the guanidino PNAs are superior to amino PNAs. The cationic PNAs were found to be specific toward their complementary DNA target as seen from their significantly lower binding with DNA having single base mismatch. The differential binding avidity of cationic PNAs was assessed by the displacement of DNA duplex intercalated ethidium bromide and gel electrophoresis. The live cell imaging of amino/guanidino PNAs demonstrated their ability to penetrate the cell membrane in 3T3 and MCF-7 cells, and cationic PNAs were found to be accumulated in the vicinity of the nuclear membrane in the cytoplasm. Fluorescence-activated cell sorter (FACS) analysis of cell permeability showed the efficiency to be dependent upon the nature of cationic functional group, with guanidino PNAs being better than the amino PNAs in both cell lines. The results are useful to design new biofunctional cationic PNA analogues that not only bind RNA better but also show improved cell permeability.

Analysis of complementarity requirements for plant microRNA targeting using a Nicotiana benthamiana quantitative transient assay.

MicroRNAs (miRNAs) guide RNA-induced silencing complexes to target RNAs based on miRNA-target complementarity. Using a dual-luciferase based sensor system in Nicotiana benthamiana, we quantitatively assessed the relationship between miRNA-target complementarity and silencing efficacy measured at both the RNA and protein levels, using several conserved miRNAs and their known target sites from Arabidopsis thaliana. We found that naturally occurring sites have variable efficacies attributable to their complementarity patterns. We also observed that sites with a few mismatches to the miRNA 3' regions, which are common in plants, are often equally effective and sometimes more effective than perfectly matched sites. By contrast, mismatches to the miRNA 5' regions strongly reduce or eliminate repression efficacy but are nonetheless present in several natural sites, suggesting that in some cases, suboptimal miRNA efficacies are either tolerated or perhaps selected for. Central mismatches fully abolished repression efficacy in our system, but such sites then became effective miRNA target mimics. Complementarity patterns that are functional in animals (seed sites, 3'-supplementary sites, and centered sites) did not reliably confer repression, regardless of context (3'-untranslated region or open reading frame) or measurement type (RNA or protein levels). Overall, these data provide a robust and empirical foundation for understanding, predicting, and designing functional miRNA target sites in plants.

Overlapping genes coded in the 3'-to-5'-direction in mitochondrial genes and 3'-to-5' polymerization of non-complementary RNA by an 'invertase'.

Suppressor tRNAs induce expression of additional (off-frame) genes coded by stopless genetic codes without lengthening genomes, decreasing DNA replication costs. RNA 3'-to-5' polymerization by tRNAHis guanylyltransferase suggests further cryptic code: hypothetical 'invertases' polymerizing in the 3'-to-5' direction, advancing in the 5'-to-3' direction would produce non-complementary RNA templated by regular genes, with different coding properties. Assuming 'invertase' activity, BLAST analyses detect GenBank-stored RNA ESTs and proteins (some potentially coding for the hypothesized invertase) for human mitochondrial genes. These peptides' predicted secondary structures resemble their GenBank homologues'. 3'-to-5' EST lengths increase with their self-hybridization potential: Single-stranded RNA degradation perhaps limits 3'-to-5' elongation. Independent methods confirm predicted 3'-to-5' overlapping genes: (a) Presumed 3'-to-5' overlapping genes avoid codons belonging to circular codes; (b) Spontaneous replicational deamination (mutation) gradients occur at 3rd codon positions, unless these are involved in overlap coding, because mutations are counter selected in overlapping genes. Tests a and b converge on predicted 3'-to-5' gene expression levels. Highly expressed ones include also fewer stops, and mitochondrial genomes (in Primates and Drosophila) adapt to avoid dependence of 3'-to-5' coding upon antitermination tRNA activity. Secondary structure, circular code, gradient and coevolution analyses yield each clear positive results independently confirming each other. These positive results (including physical evidence for 3'-to-5' ESTs) indicate that 3'-to-5' coding and invertase activity is an a priori improbable working hypothesis that cannot be dismissed. Note that RNAs produced by invertases potentially produce triple-stranded DNA:RNA helices by antiparallel Hoogsteen pairings at physiological pH, as previously observed for mitochondrial genomes.

May cyclic nucleotides be a source for abiotic RNA synthesis?

Nucleic bases are obtained by heating formamide in the presence of various catalysts. Formamide chemistry also allows the formation of acyclonucleosides and the phosphorylation of nucleosides in every possible position, also affording 2',3' and 3',5' cyclic forms. We have reported that 3',5' cyclic GMP and 3',5' cyclic AMP polymerize in abiotic conditions yielding short oligonucleotides. The characterization of this reaction is being pursued, several of its parameters have been determined and experimental caveats are reported. The yield of non-enzymatic polymerization of cyclic purine nucleotides is very low. Polymerization is strongly enhanced by the presence of base-complementary RNA sequences.

Design of a microsphere-based high-throughput gene expression assay to determine estrogenic potential.

Recently gene expression studies have been multiplied at an accelerated rate by the use of high-density microarrays. By assaying thousands of transcripts at a time, microarrays have led to the discovery of dozens of genes involved in particular biochemical processes, for example, the response of a tissue/organ to a given chemical with therapeutic or toxic properties. The next step in these studies is to focus on the response of a subset of relevant genes to verify or refine potential therapeutic or toxic properties. We have developed a sensitive, high-throughput gene expression assay for this purpose. In this assay, based on the Luminex xMAP system, carefully selected oligonucleotides were covalently linked to fluorescently coded microspheres that are hybridized to biotinylated cRNA followed by amplification of the signal, which results in a rapid, sensitive, multiplexed assay platform. Using this system, we have developed an RNA expression profiling assay specific for 17 estrogen-responsive transcripts and three controls. This assay can evaluate up to 100 distinct analytes simultaneously in a single sample, in a 96-well plate format. This system has improved sensitivity versus existing microsphere-based assays and has sensitivity and precision comparable with or better than microarray technology. We have achieved detection levels down to 1 amol, detecting rare messages in complex cRNA samples, using as little as 2.5 microg starting cRNA. This assay offers increased throughput with decreased costs compared with existing microarray technologies, with the trade-off being in the total number of transcripts that can be analyzed.

Allosteric aptamers: targeted reversibly attenuated probes.

Aptamers are unique nucleic acids with regulatory potentials that differ markedly from those of proteins. A significant feature of aptamers not possessed by proteins is their ability to participate in at least two different types of three-dimensional structure: a single-stranded folded structure that makes multiple contacts with the aptamer target and a double-helical structure with a complementary nucleic acid sequence. We have made use of this structural flexibility to develop an aptamer-based biosensor (a targeted reversibly attenuated probe, TRAP) in which hybridization of a cis-complementary regulatory nucleic acid (attenuator) controls the ability of the aptamer to bind to its target molecule. The central portion of the TRAP, between the aptamer and the attenuator, is complementary to a target nucleic acid, such as an mRNA, which is referred to as a regulatory nucleic acid (regNA) because it regulates the activity of the aptamer in the TRAP by hybridization with the central (intervening) sequence. The studies reported here of the ATP-DNA TRAP suggest that, as well as inhibiting the aptamer, the attenuator also acts as a structural guide, much like a chaperone, to promote proper folding of the TRAP such that it can be fully activated by the regDNA. We also show that activation of the aptamer in the TRAP by the complementary nucleic acid at physiological temperatures is sensitive to single-base mismatches. Aptamers that can be regulated by a specific nucleic sequence such as in an mRNA have potential for many in vivo applications including regulating a particular enzyme or signal transduction pathway or imaging gene expression in vivo.

A novel component of the ubiquitin pathway, ubiquitin carboxyl extension protein 1 is overexpressed in prostate cancer.

Prostate cancer is among the most common tumors in industrialized nations. However, little is known about the molecular events underlying its development. In the present study we used suppressive subtraction hybridization (SSH) in combination with laser-assisted microdissection in order to compare gene expression between prostate carcinoma and the normal prostate proper. Both are mixed tissues which consist of an epithelial and a stromal compartment. We first compared mRNA (cDNA) expression by SSH and then used real-time quantitative RT-PCR analysis of microdissected tissue probes in order to verify differential expression of subtracted cDNA clones. We also used differentially expressed cDNAs for the synthesis of radiolabelled riboprobes in order to attribute differential expression to specific cell types in tissue sections by in situ hybridization. Using this approach we found an up-regulation of ubiquitin carboxyl extension protein 1 (UBCEP-1) mRNA in prostate carcinoma cells compared to the normal glandular epithelium of the prostate proper. UBCEP-1 mediated ubiquitin chain elongation may promote prostate carcinoma development by increasing via the proteasome pathway the degradation of proteins which are involved in growth inhibition or apoptosis.

Substrate specificities of bacterial and human AlkB proteins.

Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.

Inhibition of telomerase activity by preventing proper assemblage.

Telomerase is a ribonucleoprotein complex that acts as a reverse transcriptase in the maintenance of chromosome ends. Because the vast majority of cancer cells require telomerase activity, telomerase has become a target for anticancer drug discovery. Here, we describe a new approach for targeting telomerase by blocking the association between the telomerase catalytic subunit, hTERT, and key elements of the human telomerase RNA subunit, hTR. By examining the effects of oligonucleotides that hybridize to various regions of hTR, we identified two regions of the RNA subunit that are sensitive to molecular interactions leading to telomerase inhibition. Oligonucleotides that hybridize to either the P3/P1 pairing region or to the CR4-CR5 domain of hTR, hTRas009, and hTRas010, respectively, inhibit telomerase activity when added to recombinant hTERT and hTR prior to assemblage. However, addition of hTRas009 or hTRas010 to preassembled telomerase resulted in little or no inhibition. We also examined the ability of hTRas009 and hTRas010 to inhibit binding of hTR and hTR fragments to hTERT. We found that hTRas009 inhibited approximately 50% of the maximum binding between the pseudoknot fragment of hTR (nucleotides 46-209) and hTERT, whereas hTRas010 inhibited over 90% of the maximum binding between the CR4-CR5 fragment of hTR (nucleotides 243-328) and hTERT. In addition, neither oligonucleotide was able to appreciably inhibit the binding of full-length hTR to hTERT, although both oligonucleotides used in conjunction decreased binding by approximately 50%. We propose that the P3/P1 pairing region and CR4-CR5 domain represent viable targets to inhibit telomerase by perturbing proper assemblage of the active complex.

A new route to 2'-O-alkyl-2-thiouridine derivatives via 4-O-protection of the uracil base and hybridization properties of oligonucleotides incorporating these modified nucleoside derivatives.

Oligonucleotides containing 2-thiouridine (s2U) in place of uridine form stable RNA duplexes with complementary RNAs. Particularly, this modified nucleoside has proved to recognize highly selectively adenosine, the genuine partner, without formation of a mismatched base pair with the guanosine counterpart. In this paper, we describe new methods for the synthesis of 2-thiouridine and various 2'-O-alkyl-2-thiouridine derivatives. Oligoribonucleotides having these modified nucleoside derivatives were synthesized, and their hybridization and structural properties were studied in detail by the 1H NMR analysis of these modified nucleosides and Tm experiments of RNA duplexes with their complementary RNA strands.

Synthesis of antisense oligonucleotides carrying modified 2-5A molecules at their 5'-termini and their properties.

The synthesis of 8-methyladenosine-substituted 2-5A tetramers with hydroxyalkyl groups at the 5'-phosphates and the corresponding 2-5A-antisense chimeras is described. These oligonucleotides were synthesized by the phosphoramidite method with a DNA/RNA synthesizer. These 2-5A tetramers with hydroxyethyl and hydroxybutyl groups at their 5'-phosphates were more resistant to hydrolysis by alkaline phosphatase than those without the hydroxyalkyl groups. Incorporation of the hydroxyethyl group into the 2-5A tetramer and 2-5A-antisense chimera slightly reduced the abilities of their analogues to activate recombinant human RNase L, but the abilities of the 2-5A tetramer and the 2-5A-antisense chimera both with the hydroxyethyl group and 8-methyladenosine returned to 80 and 50% relative to those of the oligonucleotides without the hydroxyethyl group and 8-methyladenosine, respectively. Furthermore, the enzyme activated by 8-methyladenosine-substituted 2-5A-antisense chimera with the hydroxyethyl group cleaved the complementary RNA as efficiently as that activated by 2-5A-antisense chimera without the hydroxyethyl group and 8-methyladenosine. Thus, the 2-5A-antisense chimera carrying the hydroxyethyl group and 8-methyladenosine will be a candidate for a novel antisense molecule.

Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2'F-ANA constructs containing acyclic nucleotide inserts.

The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.

Synthesis and hybridization properties of RNA containing 8-chloroadenosine.

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3'- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by approximately 5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.

Quantitative assessment of the use of modified nucleoside triphosphates in expression profiling: differential effects on signal intensities and impacts on expression ratios.

BACKGROUND: The power of DNA microarrays derives from their ability to monitor the expression levels of many genes in parallel. One of the limitations of such powerful analytical tools is the inability to detect certain transcripts in the target sample because of artifacts caused by background noise or poor hybridization kinetics. The use of base-modified analogs of nucleoside triphosphates has been shown to increase complementary duplex stability in other applications, and here we attempted to enhance microarray hybridization signal across a wide range of sequences and expression levels by incorporating these nucleotides into labeled cRNA targets. RESULTS: RNA samples containing 2-aminoadenosine showed increases in signal intensity for a majority of the sequences. These results were similar, and additive, to those seen with an increase in the hybridization time. In contrast, 5-methyluridine and 5-methylcytidine decreased signal intensities. Hybridization specificity, as assessed by mismatch controls, was dependent on both target sequence and extent of substitution with the modified nucleotide. Concurrent incorporation of modified and unmodified ATP in a 1:1 ratio resulted in significantly greater numbers of above-threshold ratio calls across tissues, while preserving ratio integrity and reproducibility. CONCLUSIONS: Incorporation of 2-aminoadenosine triphosphate into cRNA targets is a promising method for increasing signal detection in microarrays. Furthermore, this approach can be optimized to minimize impact on yield of amplified material and to increase the number of expression changes that can be detected.

2'-O,4'-C-ethylene-bridged nucleic acids (ENA): highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug.

To develop antisense oligonucleotides, novel nucleosides, 2'-O,4'-C-ethylene nucleosides and their corresponding phosphoramidites, were synthesized as building blocks. The 1H NMR analysis showed that the 2'-O,4'-C-ethylene linkage of these nucleosides restricts the sugar puckering to the N-conformation as well as the linkage of 2'-O,4'-C-methylene nucleosides which are known as bridged nucleic acids (BNA) or locked nucleic acids (LNA). The ethylene-bridged nucleic acids (ENA) showed a high binding affinity for the complementary RNA strand (DeltaT(m)=+5.2 degrees C/modification) and were more nuclease-resistant than natural DNA and BNA/LNA. These results indicate that ENA have better properties as antisense oligonucleotides than BNA/LNA.

Epstein-Barr virus complementary strand transcripts (CSTs/BARTs) and cancer.

Epstein-Barr virus (EBV) encodes a family of related transcripts, the complementary strand transcripts (CSTs) or BARTs (Bam A rightward transcripts). These are present in all types of EBV infection but are expressed to particularly high levels in nasopharyngeal carcinomas. Although convincing demonstration of protein expression from these transcripts is still subject to some debate, potential proteins encoded by them have been shown to modify Notch signalling pathways.

Theoretical design of antisense genes with statistically increased efficacy.

Endogenous expression of antisense RNA represents one major way of applying antisense nucleic acids. To express antisense RNA intracellularly, recombinant antisense genes have to be designed and introduced into cells where the target RNA is encountered. Efficient annealing between the antisense RNA and the target RNA is crucial for efficacy and is strongly influenced by RNA structure. Here we extend structural rules for the design of in vitro transcribed antisense RNAs to the design of recombinant antisense genes. Intracellularly expressed antisense RNA transcripts contain a central antisense portion and additional flanking vector-derived sequences. A computer algorithm was generated to compose large sets of antisense genes, to calculate secondary structures of the transcribed sequences and to select for favorable structures of antisense RNA in terms of annealing and efficacy. The biological test system to measure efficiency of antisense genes was human immunodeficiency virus type 1 (HIV-1) replication in 293T cells. When considering the lower intracellular steady-state levels of favorably structured endogenous transcripts, an antisense effect against HIV-1 replication was observed that was up to 60-fold stronger than that measured for predicted unfavorable species. The computational selection was successful for antisense portions of 300 nt but not 100 nt in length. This theoretical design of antisense genes supports their improved application under time- and labor-saving conditions.

Studies on anti-human immunodeficiency virus oligonucleotides that have alternating methylphosphonate/phosphodiester linkages.

Preliminary investigations of the physical properties of oligonucleotide analogs that contain alternating methylphosphonate/phosphodiester linkages are described. An alternating oligo-2'-O-methylribonucleoside methylphosphonate, oligomer 1676, whose sequence is complementary to the upper hairpin region of human immunodeficiency virus TAR RNA, has been synthesized. This 15-mer forms a very stable duplex with its complementary RNA target, whose melting temperature is 71 degrees C. Introduction of two mismatched bases reduces the melting temperature by 16 degrees C. Similar results were obtained with the all-phosphodiester version of oligomer 1676, which demonstrates that introduction of the methylphosphonate linkages does not significantly perturb the ability of the oligo-2'-O-methylribonucleoside methylphosphonate to bind to RNA. Unlike the phosphodiester oligomer, however, oligomer 1676 is completely resistant to hydrolysis by the 3'-exonuclease activity found in mammalian serum. The interactions between nuclease-resistant, 5'-psoralen-derivatized, alternating oligo-2'-deoxypyrimidine methylphosphonates and double-stranded DNA were also studied. A 15-mer that contains thymine, 5-methylcytosine, and 5-propynyl-uracil forms a triplex with a polypurine tract found in the env gene of human immunodeficiency virus proviral DNA with an apparent dissociation constant of 400 nM at 22 degrees C. Maximal triplex formation by these oligomers is observed at approximately 2.5 mM magnesium, whereas maximal triplex formation by the corresponding all-phosphodiester oligomers occurs between 10 and 20 mM magnesium. This reduced magnesium dependence most likely results from reduced charge repulsion between the backbones of the methylphosphonate oligomer and purine strand of the target. The nuclease stability and ability of the methylphosphonate oligomers to form stable complexes with their target nucleic acids suggest that these oligomers are potential candidates for use as antisense or antigene agents in cell culture.