Characterization of a Potent and Specific Class of Antisense Oligonucleotide Inhibitor of Human Protein Kinase C-α Expression

Abstract

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-α expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2′ modifications have been incorporated into the protein kinase C-α targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2′-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5° C per modification) and an increase in resistance toward both 3′-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-α in mediating the phorbol ester-induced changes in c-fos,c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-α protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos orjunB. These results demonstrate that 2′-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-α expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides. The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-α expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2′ modifications have been incorporated into the protein kinase C-α targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2′-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5° C per modification) and an increase in resistance toward both 3′-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-α in mediating the phorbol ester-induced changes in c-fos,c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-α protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos orjunB. These results demonstrate that 2′-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-α expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides. The protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; AP-1, activator protein-1; CGE, capillary gel electrophoresis; DOTMA/DOPE, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/dioleoyl phosphatidylethanolamine; GAPDH, glycerol-3-phosphate dehydrogenase; 2′-MOE, 2′-O-(2-methoxy)ethyl; P=O, phosphodiester; P=S, phosphorothioate; TPA, 12-O-tetradecanoylphorbol-13-acetate; ICAM, intracellular adhesion molecule. 1The abbreviations used are: PKC, protein kinase C; AP-1, activator protein-1; CGE, capillary gel electrophoresis; DOTMA/DOPE, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/dioleoyl phosphatidylethanolamine; GAPDH, glycerol-3-phosphate dehydrogenase; 2′-MOE, 2′-O-(2-methoxy)ethyl; P=O, phosphodiester; P=S, phosphorothioate; TPA, 12-O-tetradecanoylphorbol-13-acetate; ICAM, intracellular adhesion molecule. family of isozymes is composed of at least 11 different, but structurally related serine/threonine kinases. These can be subdivided on the basis of structural and biochemical similarities into three groups, the conventional (α,βI, βII, and γ), the novel (δ, ε, η, θ and μ), and the atypical (ζ and ι) (1Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4207) Google Scholar, 2Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (917) Google Scholar, 3Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar). The classic PKCs and the novel PKCs are activated by 1,2-diacylglycerol, which is generated by phospholipase cleavage of membrane phospholipids. These phospholipases are regulated by many growth factors and hormones, and it is therefore widely thought that PKC isozymes play an important role in regulating cell proliferation and differentiation, as well as short-term cellular responses, such as secretion and ion flux (1Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4207) Google Scholar). The identification of multiple members of the PKC family has lead to speculation that individual isozymes play different roles in regulating different cell functions (4Hug H. Sarre T.F. Biochem J. 1993; 291: 329-343Crossref PubMed Scopus (1215) Google Scholar). Much evidence is available to support this hypothesis. For example, expression profiles of the individual family members is extremely heterogeneous, both at the tissue and the subcellular levels (4Hug H. Sarre T.F. Biochem J. 1993; 291: 329-343Crossref PubMed Scopus (1215) Google Scholar, 5Buchner K. Eur. J. Biochem. 1995; 228: 211-221Crossref PubMed Scopus (159) Google Scholar). In addition, the substrate specificities of purified proteins are very different, and the responses of isozymes to stimuli differ not just between isotypes, but also between the same isotype stimulated in different cell types. Considerable effort has been made over the last 10 years to develop isozyme-specific inhibitors of PKC to allow the dissection of the PKC-dependent signaling pathways (6Hofmann J. FASEB J. 1997; 11: 649-669Crossref PubMed Scopus (331) Google Scholar). These efforts are hampered by the similarities in protein structure of the many isozymes of PKC, which make the identification of specific, small molecule enzyme inhibitors difficult. To overcome this difficulty, we have used antisense oligonucleotides to inhibit the expression of individual isozymes of PKC (7Dean N.M. McKay R. Condon T.P. Bennett C.F. J. Biol. Chem. 1994; 269: 16416-16424Abstract Full Text PDF PubMed Google Scholar, 8Dean N.M. McKay R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11762-11766Crossref PubMed Scopus (247) Google Scholar). Antisense oligonucleotides can be targeted to mRNA sequences which are unique to a given PKC isozyme, leading to the selective inhibition in expression of that isozyme (9Dean N.M. McKay R. Miraglia L. Geiger T. Muller M. Fabbro D. Bennett C.F. Biochem. Soc. Trans. 1996; 24: 623-629Crossref PubMed Scopus (40) Google Scholar, 10McGraw K. McKay R. Miraglia L. Boggs R. Pribble J.P. Muller M. Geiger T. Fabbro D. Dean N.M. Anti-Cancer Drug Design. 1997; 12: 315-326PubMed Google Scholar). The long half-lives of some PKC proteins (and other proteins which have been targeted with antisense oligonucleotides) have proven problematic, as we have found that the most widely used oligonucleotide modification available, the phosphorothioate (P=S) oligodeoxynucleotide, is metabolized in cells over time. This leads to a loss of activity over a 48–72-h period, which can make inhibition of some PKC isozymes difficult (7Dean N.M. McKay R. Condon T.P. Bennett C.F. J. Biol. Chem. 1994; 269: 16416-16424Abstract Full Text PDF PubMed Google Scholar, 8Dean N.M. McKay R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11762-11766Crossref PubMed Scopus (247) Google Scholar, 11McKay R.M. Cummins L.L. 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This should allow for the development not just of improved antisense inhibitors of PKC, but of a more generalized class of antisense effective against any mRNA target which encodes a protein with a long half-life. In the present study, the biophysical and pharmacological activity of oligonucleotides containing the recently described 2′-O-(2-methoxy)ethyl (2′-MOE) modification (20Altmann K.-H. Dean N.M. Fabbro D. Freier S.M. Geiger T. Haner R. Hüsken D. Martin P. Monia B.P. Muller M. Natt F. Nicklin P. Phillips J. Pieles U. Sasmor H. Moser H.E. Chimia. 1996; 50: 168-176Google Scholar), with both 2-O-methyl (2′-M) and 2′-deoxy containing oligonucleotides are contrasted. Antisense oligonucleotides can inhibit the expression of proteins by a number of potential mechanisms (21Dolnick B.J. Cancer Invest. 1991; 9: 185-194Crossref PubMed Scopus (44) Google Scholar, 22Crooke S.T. Annu. Rev. Pharmacol. Toxicol. 1992; 32: 329-376Crossref PubMed Scopus (414) Google Scholar, 23Wagner R.W. 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FEBS Lett. 1993; 325: 123-127Crossref PubMed Scopus (96) Google Scholar, 18Altmann K.-H. Fabbro D. Dean N.M. Geiger T. Monia B.P. Muller M. Nicklin P. Biochem. Soc. Trans. 1996; 24: 630-637Crossref PubMed Scopus (101) Google Scholar, 27Cook P.D. Anti-Cancer Drug Design. 1991; 6: 585-607PubMed Google Scholar, 28Wagner R.W. Matteucci M.D. Lewis J.G. Gutierrez A.J. Moulds C. Froehler B.C. Science. 1993; 260: 1510-1513Crossref PubMed Scopus (402) Google Scholar). This can be overcome by the inclusion of 2′-deoxy residues into an antisense oligonucleotide, in combination with 2′-alkyl modifications, in a motif that will support RNase H cleavage (chimeric oligonucleotides) (11McKay R.M. Cummins L.L. Graham M.J. Lesnick E.A. Owens S.R. Winniman M. Dean N.M. Nucleic Acids Res. 1996; 24: 411-417Crossref PubMed Scopus (63) Google Scholar, 15Monia B.P. Lesnik E.A. Gonzalez C. Lima W.F. McGee D. Guinosso C.J. Kawasaki A.M. Cook P.D. Freier S.M. J. Biol. Chem. 1993; 268: 14514-14522Abstract Full Text PDF PubMed Google Scholar, 29Inoue H. Hayase Y. Iwai S. Ohtsuke E. FEBS Lett. 1987; 215: 327-330Crossref PubMed Scopus (219) Google Scholar, 30Furdon P.J. Dominski Z. Kole R. Nucleic Acids Res. 1989; 17: 9193-9204Crossref PubMed Scopus (135) Google Scholar, 31Larrouy B. Blonski C. Boiziau C. Stuer M. Moreau S. Shire D. Toulme J.-J. Gene (Amst.). 1992; 121: 189-194Crossref PubMed Scopus (53) Google Scholar). In our studies reported here, an oligonucleotide containing 2-MOE modification in such a configuration was found to be at least 20-fold more active than conventional P=S oligodeoxynucleotides at reducing the expression of PKC-α mRNA in A549 lung carcinoma cells. This inhibition resulted in a time dependent and oligonucleotide-specific reduction in expression of PKC-α protein. This has allowed us to examine the role played by PKC-α in regulating the expression of members of the AP-1 family of transcription factors in A549 lung carcinoma cells. Activation of PKC by phorbol esters leads to an increase in expression of the fos and junfamily members, by both increased transcription and increases in mRNA stability. The PKC isoform responsible for this increase is not clear, as A549 cells express multiple phorbol ester binding PKC isozymes, including PKC-α, δ, ε, and η. Depletion of PKC-α from A549 cells had a dramatic effect on the increase in c-jun mRNA expression, reducing the up-regulation to levels seen in control cells. In contrast, the phorbol ester dependent increase in c-fos and junB mRNA expression was not inhibited. Human A549 lung carcinoma cells were obtained from the American Type Tissue Collection (ATCC). Cells were grown in Dulbecco's modified Eagle's medium containing 1 g of glucose/liter and 10% fetal calf serum and routinely passaged when 90–95% confluent. Phosphorothioate oligodeoxynucleotides were synthesized as described previously (7Dean N.M. McKay R. Condon T.P. Bennett C.F. J. Biol. Chem. 1994; 269: 16416-16424Abstract Full Text PDF PubMed Google Scholar). 2′-O-Methyl and 2′-MOE oligonucleotides were synthesized as described (32Baker B.F. Lot S.S. Condon T.P. Cheng-Flournoy S. Lesnik E.A. Sasmor H.M. Bennett C.F. J. Biol. Chem. 1997; 272: 11994-12000Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Absorbanceversus temperature profiles were performed as described previously (33Chiang M.Y. Chan H. Zounes M.A. Freier S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: 18162-18171Abstract Full Text PDF PubMed Google Scholar). Briefly, antisense oligonucleotides were hybridized to complementary RNA strands and T m values and free energies of duplex formation were obtained. Values are the averages of three experiments. A549 cells were grown to 60–70% confluence in T-75 flasks. The cells were then washed twice with Dulbecco's modified Eagle's medium and then 5 ml of Dulbecco's modified Eagle's medium containing 20 μg/ml N-[1-(2, 3-dioleyloxy)propyl]-n, n,n-trimethylammonium chloride/dioleoyl phosphatidylethanolamine (DOTMA/DOPE) (Lipofectin®)(Life Technologies, Inc.) solution was added to the flasks. Oligonucleotides were added to the required concentration from a 10 μm stock solution and the flask swirled to mix the solutions. The cells were then incubated at 37° C for 4 h and then the DOTMA/DOPE/oligonucleotide solution was aspirated off and replaced with medium for the indicated time. Oligonucleotide resistance to snake venom 3′-phosphodiesterase was determined as described previously (11McKay R.M. Cummins L.L. Graham M.J. Lesnick E.A. Owens S.R. Winniman M. Dean N.M. Nucleic Acids Res. 1996; 24: 411-417Crossref PubMed Scopus (63) Google Scholar). Briefly, the oligonucleotides were gel purified and 5′-end-labeled with high performance liquid chromatography-purified [γ-32P]ATP (ICN). The oligonucleotides (100 nm) were then incubated with snake venom phosphodiesterase (U. S. Biochemical Corp./Amersham) (5 × 10−3units/ml) for the indicated times. The oligonucleotide metabolites were then resolved on a 20% denaturing polyacrylamide gel followed by quantitation by PhosphorImager (Molecular Dynamics) analysis. A549 cells were treated with 500 nm oligonucleotides as described above and allowed to recover for 72 h. At this time, metabolites were recovered and analyzed by capillary gel electrophoresis as described previously (11McKay R.M. Cummins L.L. Graham M.J. Lesnick E.A. Owens S.R. Winniman M. Dean N.M. Nucleic Acids Res. 1996; 24: 411-417Crossref PubMed Scopus (63) Google Scholar). After digestion with proteinase K, oligonucleotide metabolites were recovered by sequential passages through an anion exchange column and a reverse phase column. Analysis of the samples by capillary gel electrophoresis was performed on a Beckman 5010 P/ACE capillary electrophoresis unit. Total mRNA was extracted from cells and resolved on agarose gels as described previously (7Dean N.M. McKay R. Condon T.P. Bennett C.F. J. Biol. Chem. 1994; 269: 16416-16424Abstract Full Text PDF PubMed Google Scholar). These were transferred to nylon membrane (Bio-Rad) and probed with 32P-radiolabeled cDNA probes for different PKC isozymes (7Dean N.M. McKay R. Condon T.P. Bennett C.F. J. Biol. Chem. 1994; 269: 16416-16424Abstract Full Text PDF PubMed Google Scholar). Additionally, gels were probed with [32P]cDNA probes for c-fos, c-jun, junB (ATCC). Gels were routinely stripped and reprobed with radiolabeled human glycerol-3-phosphate dehydrogenase (GAPDH) probe to confirm equal loading. Radioactive bands were quantitated using a PhosphorImager, and typically we measure only the upper of the two PKC-α transcripts, although both are reduced with identical kinetics upon oligonucleotide treatment of cells. 2R. A. McKay, L. J. Miraglia, L. L. Cummins, S. R. Owens, H. Sasmor, and N. M. Dean, unpublished observation. PKC isozyme protein expression was determined by Western blotting (7Dean N.M. McKay R. Condon T.P. Bennett C.F. J. Biol. Chem. 1994; 269: 16416-16424Abstract Full Text PDF PubMed Google Scholar). The antibodies used were obtained as indicated. PKC-α, UBI; PKC-δ, Santa Cruz Biotechnology; PKC-ε, a gift from Dr. Doriano Fabbro, Novartis Pharmaceuticals; PKC-η, BioMol; PKC-μ, Santa Cruz Biotechnology; and PKC-ζ (UBI). A549 cells were treated with oligonucleotides for 3 days. Cells were then washed in cold phosphate-buffered saline, scraped, and pelleted into a sample preparation buffer (50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 10 mm EGTA, 50 mm2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 10 mm benzamidine). A cytosolic fraction was prepared by centrifugation at 100,000 × g for 1 h at 4° C. PKC enzyme activity was determined by measuring the ability of the cytosolic protein extract to phosphorylate a synthetic peptide substrate in the absence or presence of phosphatidylserine in an enzyme-linked immunosorbent-based assay according to the manufacturers instructions (MBL Co. Ltd., Nagoya, Japan). The final concentrations of the reaction mixture used were 25 mm Tris-HCl, pH 7.0, 3 mm MgCl2, 0.1 mm ATP, 2 mm CaCl2, 0.5 mm EDTA, 1 mm EGTA, 5 mm 2-mercaptoethanol, ±50 μg/ml phosphatidylserine. PKC activity is defined as phosphatidylserine-dependent kinase activity. The inhibition of PKC-α mRNA expression by the uniform phosphorothioate oligodeoxynucleotide sequence used here is believed to be mediated by RNase H (7Dean N.M. McKay R. Condon T.P. Bennett C.F. J. Biol. Chem. 1994; 269: 16416-16424Abstract Full Text PDF PubMed Google Scholar). The 2′-modifications examined in the present study do not form substrates for RNase H, and therefore need to be incorporated into the oligonucleotide in combination with oligodeoxynucleotide residues to effect this mechanism of mRNA degradation (18Altmann K.-H. Fabbro D. Dean N.M. Geiger T. Monia B.P. Muller M. Nicklin P. Biochem. Soc. Trans. 1996; 24: 630-637Crossref PubMed Scopus (101) Google Scholar) (20Altmann K.-H. Dean N.M. Fabbro D. Freier S.M. Geiger T. Haner R. Hüsken D. Martin P. Monia B.P. Muller M. Natt F. Nicklin P. Phillips J. Pieles U. Sasmor H. Moser H.E. Chimia. 1996; 50: 168-176Google Scholar, 34Dean N.M. Griffey R.H. Antisense & Nucleic Acid Drug Dev. 1997; 7: 229-233Crossref PubMed Scopus (63) Google Scholar). The number of contiguous oligodeoxynucleotide residues required in an oligonucleotide to support RNase H cleavage of a hybridized RNA have been proposed to range from 3 to 8 (15Monia B.P. Lesnik E.A. Gonzalez C. Lima W.F. McGee D. Guinosso C.J. Kawasaki A.M. Cook P.D. Freier S.M. J. Biol. Chem. 1993; 268: 14514-14522Abstract Full Text PDF PubMed Google Scholar, 31Larrouy B. Blonski C. Boiziau C. Stuer M. Moreau S. Shire D. Toulme J.-J. Gene (Amst.). 1992; 121: 189-194Crossref PubMed Scopus (53) Google Scholar, 35Quartin R.S. Brakel C.L. Wetmur J.G. Nucleic Acids Res. 1989; 17: 7253-7262Crossref PubMed Scopus (64) Google Scholar, 36Boiziau C. Larrouy B. Moreau S. Cazenave C. Shire D. Toulm J.-J. Biochem. Soc. Trans. 1992; 20: 764-768Crossref PubMed Scopus (10) Google Scholar, 37Giles R.V. Tidd D.M. Anti-Cancer Drug Design. 1992; 7: 37-48PubMed Google Scholar, 38Lima W.F. Crooke S.T. J. Biol. Chem. 1997; 272: 27513-27516Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 39Lima W.F. Mohan V. Crooke S.T. J. Biol. Chem. 1997; 272: 18191-18199Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). To determine the requirements for this oligonucleotide sequence, we have initially incorporated 4, 6, or 8 contiguous oligodeoxynucleotide residues (deoxy gap) into the center of a full phosphorothioated 2′-O-methyl oligonucleotide, and determined the ability of these oligonucleotides to reduce PKC-α mRNA expression in human lung A549 cells at 500 nm concentration. As shown previously, a fully 2′-O-methyl modified compound was unable to reduce PKC-α mRNA expression (Fig. 1, Aand B). However, increasing the number of oligodeoxynucleotide residues (deoxy gap) present in the oligonucleotide resulted in a progressive increase in the ability of the sequence to reduce PKC-α mRNA expression. A contiguous stretch of 8 residues a than reduction in expression of PKC-α mRNA (Fig. 1, A A series of oligonucleotides were synthesized with 8 contiguous oligodeoxynucleotide residues with 2′-O-methyl or The of oligonucleotide was oligodeoxynucleotide for synthetic P=S were in the oligodeoxynucleotide sequence of the oligonucleotide to resistance to in this of the molecule (15Monia B.P. Lesnik E.A. Gonzalez C. Lima W.F. McGee D. Guinosso C.J. Kawasaki A.M. Cook P.D. Freier S.M. J. Biol. Chem. 1993; 268: 14514-14522Abstract Full Text PDF PubMed Google Scholar). The sequences were also prepared with phosphorothioate and The incorporation of a of 11 2′-O-methyl residues increased oligonucleotide affinity toward a complementary mRNA from to C and C as P=S or The 2′-MOE a increase in T to and and affinity of the oligonucleotides used in the present 20-mer antisense oligonucleotide targeted to sequences in the human PKC-α mRNA were synthesized with the indicated modifications The center of the antisense oligonucleotides was oligodeoxynucleotide and The were synthesized with phosphorothioate between or between In addition, the phosphorothioate oligodeoxynucleotide and a oligodeoxynucleotide were Oligonucleotide sequence is shown For m antisense oligonucleotides were hybridized to complementary RNA strands and T and free energies of duplex formation were obtained as described Values are the averages of three experiments. in a The 20-mer antisense oligonucleotide targeted to sequences in the human PKC-α mRNA were synthesized with the indicated modifications The center of the antisense oligonucleotides was oligodeoxynucleotide and The were synthesized with phosphorothioate between or between In addition, the phosphorothioate oligodeoxynucleotide and a oligodeoxynucleotide were Oligonucleotide sequence is shown For m antisense oligonucleotides were hybridized to complementary RNA strands and T and free energies of duplex formation were obtained as described Values are the averages of three experiments. The resistance of the oligonucleotides shown in was determined using a number of different in assay was used to examine the ability to withstand digestion by a snake venom phosphodiesterase which resulted in digestion of the P=S oligodeoxynucleotide time with the of a compound was (Fig. In contrast, only of the oligonucleotide was that with a this modification resistance than that obtained by a P=S oligodeoxynucleotide the two residues were in the of a P=S of (Fig. The 2′-MOE was digestion for the of the were also performed to the effects of the 2′-O-methyl and 2′-MOE modifications as P=S on oligonucleotide in tissue cells. A549 cells were treated with oligonucleotides nm in the presence of and the oligonucleotide metabolites extracted from cells 72 h and resolved by capillary gel At this time, of had with the of by in the of metabolites (Fig. of the 2′-O-methyl containing oligonucleotide was also not In contrast, of 2′-MOE modified was found (Fig. of the P=S oligodeoxynucleotide into A549 cells results in a reduction in PKC-α mRNA expression after h (7Dean N.M. McKay R. Condon T.P. Bennett C.F. J. Biol. Chem. 1994; 269: 16416-16424Abstract Full Text PDF PubMed Google Scholar). The for this reduction was In the of a P=S the 2′-O-methyl and 2′-MOE containing oligonucleotides demonstrate a and increase in and as a of the affinity of these two In the of a the 2′-O-methyl oligonucleotide is this molecule has a T m than the P=S oligodeoxynucleotide In contrast, the containing 2′-MOE compound is more active than (Fig. 4, A and B). PKC-α protein has a very long C. U. R. Fabbro D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: PubMed Scopus (54) Google Scholar) and therefore to reduce expression of this protein should oligonucleotide activity for at least three half-lives of the protein. The ability of the 2′-MOE modification to withstand digestion us to determine oligonucleotides containing these modifications reduce expression of PKC-α mRNA for of time. Oligonucleotides and well as two control oligonucleotides) were into A549 cells and PKC-α mRNA expression determined 72 h At this time is