Phosphodiester Oligodeoxynucleotides Research Articles

Specific Inhibition of Nitric Oxide Production in Macrophages by Phosphorothioate Antisense Oligonucleotides

□ The effects of antisense oligonucleotides (ODNs) on nitric oxide (NO) production induced by lipopolysaccharide (LPs) were investigated using thioglycollate-induced mouse peritoneal macrophages. Antisense phosphorothioate ODNs (S-oligo) corresponding to a sequence in the neighborhood of the AUG initiation codon of a mouse inducible nitric oxide synthase (iNOS) mRNA, which has a G-quartet motif in its antisense sequence, inhibited NO induction in a dose-dependent manner. Antisense phosphodiester ODNs (D-oligo), 5′- and 3′-terminal phosphorothioate-modified antisense ODNs and control scramble and missense S-oligos had no such effect. In addition, control nonsense and two mismatched S-oligos, which include G-quartet motif in their sequences, inhibited NO induction to ~50% of those in the control. Antisense S-oligo showed the inhibitory effect on NO production by exposure of macrophages to various concentrations of LPS. Western blot analysis using anti-mouse inducible nitric oxide synthase (iNOS) antibody revealed that antisense S-oligo specifically removed an immunoreactive band at 130kDa. In addition, the results of reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effect originated from a specific reduction of the targeted iNOS mRNA by hybridization with the antisense S-oligo. Furthermore, no ODNs affected β-actin mRNA and tumor necrosis factor α (TNF- α) expression in macrophages stimulated by LPS. These findings demonstrated that antisense S-oligo inhibited NO production derived from iNOS expression in macrophages by an antisense mechanism, including the aptameric effect partially mediated by the G-quartet motif.

Beta-Cyclodextrin derivatives as carriers to enhance the antiviral activity of an antisense oligonucleotide directed toward a coronavirus intergenic consensus sequence.

SummaryThe ability of cyclodextrins to enhance the antiviral activity of a phosphodiester oligodeoxynucleotide has been investigated. Aff18-mer oligodeoxynucleotide complementary to the initiation region of the mRNA coding for the spike protein and containing the intergenic consensus sequence of an enteric coronavirus has been tested for antiviral action against virus growth in human adenocarcinoma cells. The phosphodiester oligodeoxynucleotide only showed a limited effect on virus growth rate (from 12 to 34% viral inhibition in cells treated with 7.5 to 25 μM oligodeoxynucleotide, respectively, at a multiplicity of infection of 0.1 infectious particle per cell). In the same conditions, the phosphorothioate analogue exhibited stronger antiviral activity, the inhibition increased from 56 to 90%. The inhibitory effect of this analogue was antisense and sequence-specific. Northern blot analysis showed that the sequence-dependent mechanism of action appears to be the inhibition of mRNA transcription. We conclude that the coronavirus intergenic consensus sequence is a good target for an antisense oligonucleotide antiviral action. The properties of the phosphodiester oligonucleotide was improved after its complexation with cyclodextrins. The most important increase of the antiviral activity (90% inhibition) was obtained with only 7.5 μM oligonucleotide complexed to a cyclodextrin derivative, 6-deoxy-6-S-β-D-galactopyranosyl-6-thio-cyclomaltoheptaose in a molar ratio of 1:100. These studies suggest that the use of cyclodextrin derivatives as carrier for phosphodiester oligonucleotides delivery may be an effective method for increasing the therapeutic potential of these compounds in viral infections.

Site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides and their thermal stability and nuclease-resistance properties.

We report here the site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides (ODNs). ODNs containing both 5-( N-aminohexyl)-carbamoyl-2'-deoxyuridine (H), which serves as a tether for the further conjugation of functional groups, and 5-(N,N-dimethylaminohexyl)carbamoyl-2'-deoxyuridine (D), which contributes to the thermal stability of the duplex and to the resistance to nucleolytic hydrolysis by nucleases, were synthesized. Functional groups such as folic acid and palmitic acid were site-specifically introduced into the terminus of the aminohexyl-linker of H. The thermal stability and resistance toward nuclease digestion of the modified ODNs were studied. We found that ODNs containing D and H formed stable duplexes with both the complementary DNA and RNA strands even when a bulky functional group such as folic acid, palmitic acid or cholesterol was attached to the terminus of the amino-linker. We also found that ODN analogues which contained D were more resistant to nucleolytic degradation by exo- and endonuclease than the unmodified ODN. Furthermore, duplexes formed by ODNs containing D and the complementary RNA could elicit RNase H activity.

Effects of Oligodeoxynucleotides on the Physicochemical Characteristics and Cellular Uptake of Liposomes

We studied the effects of a phosphodiester oligodeoxynucleotide (ODN), which is antisense to the site in the neighborhood of the AUG initiation codon of the mouse tumor necrosis factor α gene (TNF-α), on the physicochemical characteristics and the cellular association of three types of liposomes with different surface charges. The physicochemical characteristics of the liposomes changed after adding ODN. When the ODN/lipid molar ratio was ~0.15 in cationic (TMAG) liposomes [consisting of N-(α-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG), dilauroylphosphatidylcholine (DLPC), and dioleolylphosphatidylethanolamine (DOPE) in a 1:2:2 ratio], but not in neutral and negatively charged liposomes, then the liposomes aggregated and fused. At higher molar ratios, these changes in TMAG liposomes were not evident. In addition, ODN inverted the ζ-potential of TMAG liposomes from positive to negative at an ODN/lipid molar ratio of ~0.15. Therefore, the aggregation and fusion induced by ODN could be explained by a lower surface charge repulsion between TMAG liposomes. On the other hand, the association of ODN with RAW264.7 cells, a mouse macrophage-like cell line, was very slight. The cellular association of ODN was significantly enhanced compared with neutral and negatively charged liposomes by encapsulation in TMAG liposomes. The ODN added to liposome suspensions did not affect the rate and extent of TMAG liposome cellular association, even at an ODN/lipid molar ratio of ~0.15. These results indicate that the lipid composition and ODN/lipid molar ratio are critical for the physicochemical characteristics of cationic liposomes. However, the changes had less influence on the cellular uptake properties of cationic liposomes.

Inhibition of troponin C production without affecting other muscle protein synthesis by the antisense oligodeoxynucleotide.

The effect of blocking expression of a specific gene with antisense phosphodiester oligodeoxynucleotides on the coordinate regulation of myogenesis was studied. Different regions of both fast and slow troponin C (TnC) mRNAs were targeted for binding of the antisense oligomer. The 5'-cap region of both mRNAs was found to be the most effective target for inhibiting the expression of these genes. Approximately 40%-60% inhibition of expression of a specific isoform of TnC was achieved. However, inhibition of the TnC expression did not appreciably alter the pattern of myogenesis of mouse C2C12 cells. The differentiated murine muscle cells were able to cope with this reduced level of the target gene expression by antisense phosphodiester oligomers. We have also used a phosphorothioate oligomer targeted against a common sequence within the coding region of both fast and slow TnC mRNAs. This oligomer was found to be ineffective in blocking TnC gene expression.

Two Neuropeptide Y (NPY) Antisense Phosphodiester Oligodeoxynucleotides Are Ineffective in Inhibiting NPY Gene Expression in the Rat Hypothalamus

Two Neuropeptide Y (NPY) Antisense Phosphodiester Oligodeoxynucleotides Are Ineffective in Inhibiting NPY Gene Expression in the Rat Hypothalamus

Rapid sequence-independent cellular response to oligodeoxynucleotides

The presence of receptors for oligodeoxynucleotides (OdN) on the surface of L929 cells has previously been described. To study the possible coupling of the receptor to cellular signal transducing systems, the effect of phosphodiester OdN of different sequences on cellular phospholipase C and protein kinase C (PKC) activities in L929 fibroblasts was studied. Treatment of cells with OdN induced an increase in 32P labeling of phosphatidic acid which was accompanied by a gradual decrease in diacylglycerol. These effects seem to be independent of the OdN sequence. PKC activity in membranes isolated from OdN-treated cells was found to be lower than that in membranes of control cells. SDS-PAGE of the 32P-labeled cellular proteins revealed that OdN treatment caused a decrease in phosphorylation of the 26 and 73 kDa cellular proteins in the cells.

Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED 100 dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the B max of [ 3H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832±39 fmol/mg protein) controls without any significant change in K D (33 nM). A corresponding reduction of [ 3H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [ 3H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.

Oligonucleotide adsorption to artificial surfaces

A necessary step in the development of a candidate drug involves characterization of the drug's interactions with the natural and synthetic surfaces it encounters. In this preliminary study, the time-, solvent-, surface-, and cation-dependency of oligodeoxynucleotide (ON) adsorption was examined by measuring loss of a radiolabeled phosphodiester ON from either cell growth media or distilled, deionized water (with or without different added cations) as a function of time and surface. As expected, ON adsorption was dependent on all the factors examined. Either silica coating of surfaces or the addition of cations, EDTA or heat-inactivated serum, may be useful techniques for reducing ON adsorptive losses.

Antisense therapy.

The sequence specificity of the antisense technique makes it an attractive basis for novel molecular therapeutics. Inhibition of gene expression by antisense in cell culture models has provided a strong rationale for identification and validation of disease targets. Analogues modified from normal phosphodiester oligodeoxynucleotides have entered clinical trials of diseases including AIDS, cancer, and inflammation. It is becoming increasingly apparent that these drugs act by means of a complex mechanism of action, and some of their effects can be sequence independent. Nevertheless, these oligodeoxynucleotides offer considerable promise as novel molecular drugs. Harnessing the therapeutic potential of this powerful technique depends on elucidation of the complex mechanism of action so that effective and meaningful therapeutic modalities can be realized.

Cellular uptake and localization of liposomal-methylphosphonate oligodeoxynucleotides.

Nuclease digestion and intracellular delivery are major factors limiting the potential use of oligodeoxynucleotides as antisense molecules. Structural analogues of phosphodiester oligodeoxynucleotides, such as phosphorothioates and methylphosphonates, are resistant to nuclease degradation and can still bind to their mRNA targets. However, their limited ability to escape from the endosomal/lysosomal compartments and reach the intracellular sites of action have dampened their potential clinical application. To circumvent this problem we have incorporated methylphosphonate oligodeoxynucleotides into liposomes. We found that the level of uptake of liposome-incorporated methylphosphonate oligodeoxynucleotides is time and concentration dependent. Maximal up take occurred at 8 h when 4-8 microM liposome-incorporated methylphosphonate oligodeoxynucleotides was added. Approximately 50% of liposome-incorporated methylphosphonate oligodeoxynucleotides were retained in cells after 24 h of incubation. Using fluorescent microscopy, intracellular fluorescence could be seen within 2.5 h of incubation. Diffused fluorescence was found throughout the cytoplasm, suggesting that the liposome-incorporated methylphosphonate oligodeoxynucleotides were not confined within the endosomal/lysosomal structures. We conclude that liposomes can effectively deliver methylphosphonate oligodeoxynucleotides to the cytoplasm, which is the major intracellular site of action for translational arrest.

Native oligodeoxynucleotides specifically active against human immunodeficiency virus type 1 in vitro: a G-quartet-driven effect?

Among a series of unmodified phosphodiester (PO)-oligodeoxynucleotides (PO-ODNs) complementary to some of the human immunodeficiency virus type 1 (HIV-1) regulatory genes, several PO-ODN sequences complementary to the vpr gene (PO-ODNs-a-vpr, where a-vpr is the antisense vpr sequence) emerged as potent inhibitors (at concentrations of 0.8 to 3.3 microM) of HIV-1 multiplication in de novo infected MT-4 cells, while they showed no cytotoxicity for uninfected cells at concentrations up to 100 microM. Unlike phosphorothioate counterparts, PO-ODN-a-vpr sequences were not inhibitory to HIV-2 multiplication in de novo infected C8166 cells and neither prevented the fusion between chronically infected and bystander CD4+ cells nor inhibited the activity of the HIV-1 reverse transcriptase in enzyme assays. Moreover, they were not inhibitory to HIV-1 multiplication in chronically infected cells. Delayed addition experiments showed that PO-ODNs-a-vpr inhibit an event in the HIV-1 replication cycle following adsorption to the host cell, but preceding reverse transcription. Structure-activity relationship studies indicated that the antiviral activity of the test PO-ODN-a-vpr sequences is not related to an antisense mechanism but to the presence, within the active sequences, of contiguous guanine residues. Physical characterization of the test PO-ODNs suggested that the active structure is a tetramer stabilized by G quartets (i.e., four G residues connected by eight hydrogen bonds).

Comparison of different antisense oligonucleotides against the BCR/ABL junction in chronic myelogenous leukemia

The activity of the BCR/ABL hybrid gene is associated with a growth advantage of the chronic myelogenous leukemia (CML) stem cell. Suppression of BCR/ABL hybrid gene expression can be a valuable tool for leukemic cell purging. Antisense oligonucleotides (ODNs) have the capacity to specifically downregulate gene expression. Data reported on the effect they have on BCR/ABL hybrid gene expression are controversial. We present data illustrating that prolongation of ODN half-life by means of chemical or sequence modification has only limited specific growth suppressive effect on BCR/ABL-positive clonogenic cells in vitro. Compared to unmodified phosphodiester ODNs (PO-ODNs) spanning the BCR/ABL junctions, modified ODNs with either a 3'-GC-clamp (GC-ODNs) or ODNs with one 3'-inverted nucleotide (3'-3' ODNs) to prevent 3'-exonuclease degradation, showed significantly prolonged extra- and intracellular half lives and different subcellular distributions in CML cell lines. In clonogenic assays from patients with CML, the modified ODNs were to some extent able to reduce colonies expressing BCR/ABL (GC-ODNs >3'-3'-ODNs >PO-ODNs). This difference did not become significant statistically. We demonstrate a substantially diminished hybridization efficacy of the modified antisense ODNs used, which may serve as a possible explanation for the failure to augment the leukemic cell purging efficacy.

Multiple mechanisms may contribute to the cellular anti-adhesive effects of phosphorothioate oligodeoxynucleotides.

Phosphorothioate oligodeoxynucleotides complementary to the p65 (Rel A) subunit of the NF-kappaB nuclear transcriptional regulatory factor have been suggested to be sequence specific blockers of cellular adhesion. We studied the effects of Rel A antisense, Rel A sense and other phosphorothioate oligodeoxynucleotides on cellular adhesion and found that blockade of adhesion was predominately non-sequence specific. Phosphorothioate oligodeoxynucleotides bind to the extracellular matrix (ECM) of NIH 3T3 cells, and to the ECM elements laminin and fibronectin. By use of a gel mobility shift assay, the association of the A subunit of laminin with a probe 12mer phosphodiester oligodeoxynucleotide could be demonstrated. This interaction was described by a single-site binding equation (K d = 14 microM). Human Rel A antisense and sense oligodeoxynucleotides, and two synthetic persulfated heparin analogs were excellent competitors of the binding of the probe oligodeoxynucleotide to laminin. Taken together, these data indicate that oligodeoxynucleotide binding occurred at or near the heparin-binding site. Competition for 5' 32p- SdT18 (an 18mer phosphorothioate homopolymer of thymidine) binding to fibronectin with the discrete heparin analogs, as well as with SdC28, was also observed. Phosphorothioate oligodeoxynucleotides (Rel A antisense >> Rel A sense) inhibited the binding of laminin to bovine brain sulfatide, but not to its cell surface receptors on MCF-7 cells. By flow cytometric analysis we have also shown, in contrast to what was observed with laminin, that phosphorothioates a non-specifically block the specific binding of fluoresceinated fibronectin to its cell surface receptors on phorbol-12,13-myristate acetate treated Jurkat cells. Blockade of specific binding occurred in the oligodeoxynucleotide treated cells in the presence or absence of oligomer in the media.

Calcium-dependent oligonucleotide cellular uptake

Oligodeoxynucleotides (ONs) are currently being tested in clinical trials as anti-viral and thrombolytic agents. Although ONs are biologically active in almost every cell line examined, these molecules are inefficiently internalized. In addition, the mechanism of ON transport to the active site in either the cytoplasm or nucleus remains ill-defined. Thus, elucidation of the mechanism of ON cellular uptake is necessary for the rational design of optimization of ON cellular uptake, delivery, and targeting. Previous studies in this laboratory have examined the mechanism of ON cellular uptake using a phosphodiester ON which is both biologically active and internalized by the Rauscher Red 5-1.5 mouse erythroleukemia cell line. The uptake is calcium-dependent since uptake is significantly reduced in calcium-free media and enhanced up to 20-fold when incubated in elevated calcium concentrations by a mechanism which may differ from uptake in physiologic calcium. Therefore, the goal of this paper is to further examine the mechanism of calcium-dependent phosphodiester ON uptake in Rauscher cells. First, to determine whether changes in intracellular free calcium [Ca] i may influence ON uptake, [Ca], was either increased or decreased prior to addition of the ON. Second, to determine whether protein kinase C or calmodulin are involved in uptake, the activity of each of these proteins was either increased or decreased. Neither altering [Ca] i or the activity of protein kinase C or calmodulin altered the extent of ON uptake in these cells. Finally, the effect of other cations were tested for their ability to increase ON uptake. Lanthanum and cadmium increased uptake whereas sodium and magnesium had no effect. These findings suggest that cation-mediated ON cellular uptake may not result from alteration of a calcium-dependent biological process.

Polydeoxyguanine motifs in a 12-mer phosphorothioate oligodeoxynucleotide augment binding to the v3 loop of HIV-1 gp120 and potency of HIV-1 inhibition independency of G-tetrad formation.

Phosphorothioate oligodeoxynucleotides belong to a class of polyanions that bind to the third variable domain (v3) of HIV-1 gp120 and inhibit infectivity of a wide variety of HIV-1 isolates. This potent v3 binding of phosphorothioate oligodeoxynucleotides, which is relatively independent of the nucleotide sequence of the oligodeoxynucleotides, decreases with chain length (below 18-mers) and is low for 8-mers. However, recent studies have observed a nucleotide sequence-dependent augmentation of phosphorothioate oligodeoxynucleotide binding to v3 for 8-mers that contain the S-dG4 motif (e.g., SdT2G4T2) and have suggested that formation of quadruple helical tetraplexes (G-tetrads) is associated with the acquisition of v3 binding ability by small phosphorothioate oligodeoxynucleotides. In the current study, a series of SdG4-containing oligodeoxynucleotides were synthesized with varying tandem length (including the 8-mer SdT2G4T2, the 12-mer SdG4T4G4, and the 28-mer SdG4(T4G4)3) and compared with phosphorothioate oligodeoxynucleotides (with similar lengths or related sequences) for (1) their inhibition of the binding of mAb 9284, which binds to the N-terminal portion of the v3 loop, (2) the values of Kc when these compounds are used as competitors of the rgp120-binding of an alkylating phosphodiester oligodeoxynucleotide probe, and (3) inhibition of HIV-1 infectivity in a cell-cell transmission model. The presence of S-dG4 motifs and the number of tandem motifs augmented v3 binding and anti-HIV-1 infectivity for small (8-mer or 12-mer oligodeoxynucleotides) but did not significantly augment the potency of 28-mers. Whereas tetraplex formation of SdT2G4T2 may contribute to its v3 binding, the 12-mer SdG4T4G4 does not migrate as the tetraplex on nonreducing gels, suggesting that S-dG4 motifs may augment anti-HIV activity by multiple mechanisms.

The therapeutic potential of antisense oligonucleotides.

Specific inhibition of gene expression by antisense agents provides the basis for rational drug discovery based on molecular targets. Due to the specificity of Watson-Crick base-pair hybridization, antisense oligodeoxynucleotides have been used extensively in attempts to inhibit gene expression in both in vitro and in vivo models. Analogues modified from normal phosphodiester oligodeoxynucleotides have entered clinical trials against diseases including AIDS and cancer. Although the precise mechanism of action of these drugs has not been clarified, these oligodeoxynucleotides offer considerable promise as novel molecular therapeutics. We review the recent attempts to harness the therapeutic potential of these oligodeoxynucleotides and appraise the near-term prospects for antisense technology.

Cellular uptake of intracerebrally administered oligodeoxynucleotides in mouse brain

Intracerebral diffusion, cellular uptake and intracellular localization of oligodeoxynucleotides (ODN) after their microinjection in mouse brains were examined. Using either tetramethylrhodamine-5-(and -6)-isothiocyanate (TRITC)- or γ- 33P ATP-labeled ODNs, it was found that both phosphodiester ODNs (D-ODN) and phosphorothioate ODNs (S-ODN) quickly diffused (up to about 500 μm) and were taken up by many cells around the injection site as early as 15 min after administration. Fluorescence labeling intensity and silver grain accumulation of D-ODNs were greatly reduced by 4 h after injection, whereas those of S-ODNs were stable beyond at least 8 h after injection. Most of labeled ODNs were found in neuronal cells as identified by immunocytochemistry for neurofilament, NF 200, and to a much lesser extent in astrocytic cells as identified by immunocytochemistry for glial fibrillary acidic protein.

Central α2-adrenoceptors and blood pressure regulation in the rat

The role of α 2-adrenoceptors in the central regulation of blood pressure has been questioned, since drugs such as clonidine stimulate both α 2-adrenoceptors and imidazoline-preferring receptors. The present work was undertaken to study the influence of α 2 D -adrenoceptors, encoded by the RG20 gene, on blood pressure in the rat. An antisense phosphodiester oligodeoxynucleotide, directed at nucleotides 4–21 of the RG20 gene, was injected in the right lateral cerebral ventricle, causing an increase in systolic blood pressure, both at 1 and 2 days after the injection, when compared to groups of control rats (injected with the sense oligodeoxynucleotide or a missense oligodeoxynucleotide or distilled water). Antisense oligodeoxynucleotides directed either at nucleotides 65–82 of the RG10 gene (encoding for α 2 C -adrenoceptors) or at nucleotides 26–43 of the RNG gene (encoding for α 2 B -adrenoceptors), failed to produce any change in blood pressure after being injected. We conclude that the α 2-adrenoceptor encoded by the RG20 gene may play a role in blood pressure regulation in the rat.

Expression of developmentally specific isoforms on non-NMDA mRNAs in the hippocampus of schizophrenics

In order to investigate the process of gene expression of N-methyl-d-aspartate glutamate receptor (NMDAR) subunits in the rat neostriatum and how this relates to motor behaviors, a single dose of antisense phosphodiester oligodeoxynucleotide specific for NMDAR1 was unilaterally applied in the neostriatum in a stereotaxic apparatus. After one day of antisense treatment, ipsilateral rotation behaviors that were induced by apomorphine were found in the treated animals. Reductions in the levels of expression of NMDAR1 and NMDAR2A messenger RNAs (NMDAR1: 20.6%; NMDAR2A: 19.7%) were found in the antisense-treated striatal tissues by reverse transcriptase–polymerase chain reaction. There was no change in the levels of NMDAR2B, NMDAR2C and NMDAR2D messenger RNAs. After two days, western blotting experiments showed that there were decreases in the levels of expression of NMDAR1 (decreased 27.6%) and NMDAR2A (decreased 19.2%) proteins in the NMDAR1 antisense-treated striatal tissues. In addition, NMDAR1 immunoreactivity was found to decrease in intensity in the NMDAR1 antisense-treated neostriatum. At the cellular level, the intensity of NMDAR1 immunoreactivity in perikarya of presumed medium spiny neurons was found to decrease.These results indicate that a single dose of NMDAR1 antisense modifies the expression of NMDAR1 messenger RNA and protein in neurons in the neostriatum. The modification in the expression of NMDAR1 has differential effects in the expression of NMDAR2 subunits. Gene expression of the native NMDAR subunits is likely to be a dynamic process. The change in gene expression of the NMDAR subunits in the neostriatum may have a profound effect on the motor behaviors of rats.