770
Bioconjugate Chem. 2001, 12, 770−775
A Ribonuclease H-Oligo DNA Conjugate That Specifically Cleaves Hepatitis B Viral Messenger RNA Cherie M. Walton, Catherine H. Wu, and George Y. Wu* Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington, Connecticut 06032. Received February 15, 2001; Revised Manuscript Received June 15, 2001
Ribonuclease H (RNaseH) recognizes and efficiently cleaves the RNA strand of DNA-RNA hybrids, but has no inherent sequence selectivity. However, the formation of DNA-RNA hybrids does require specific sequence recognition. On the basis of this concept, we wondered whether antisense oligonucleotides complementary to target RNA covalently linked to RNase H could be used to direct specific cleavage events mediated by RNase H. The aim of this research was to couple a DNA oligonucleotide to RNase H to confer specificity of ribonuclease activity toward hepatitis B viral (HBV) mRNA. A modified 13-base oligonucleotide that is specific for the DR1 region of HBV mRNA was conjugated to modified E. coli RNase H using a water soluble cross-linker. A 1200 base fragment of HBV RNA including the DR1 region was synthesized as a substrate using T7 RNA polymerase. Incubation of the RNase H-oligonucleotide conjugate with the RNA transcript resulted in cleavage of the HBV mRNA transcript in a concentration dependent manner. Eighty-five percent of substrate was cleaved under optimal conditions. Controls consisting of RNase H alone, oligonucleotide alone, and incubation of the conjugate with an unrelated mRNA substrate resulted in no cleavage activity. RNase H coupled to an HBV antisense oligonucleotide can specifically cleave target HBV transcripts.
INTRODUCTION
Ribonuclease H (RNase H) is an enzyme found in a variety of organisms ranging from viruses to mammalian cells and functions as an endonuclease that can recognize and cleave the RNA strand of a DNA-RNA hybrids (14). E. coli RNase H1 is one of the best characterized (3, 5). It requires divalent cations and generates products with 5′-phosphate and 3′-hydroxyl termini (6, 7). However, RNase H activity lacks sequence specific recognition (1-3). Coaddition of antisense molecules with the enzyme has been used to direct specificity to RNase H cleavage (8-11). However, to be biologically useful, this requires that the antisense oligos and enzyme be present in the same intracellular compartment as the target and in effective concentrations. With this goal in mind, the aim of this research was to determine whether an antisense oligo DNA could be chemically coupled to RNase H to specifically target RNase H activity to the 3′ direct repeat 1 (DR-1) region of the HBV1 mRNA. MATERIALS AND METHODS
RNase H Purification. An RNase H overproducer mutant strain (rnh gene transformed into MIC 1066, an RNase H deficient strain of E coli) was a gift from Robert Crouch, NIH. RNase H was purified according to the method of Ma et al. (1). In brief, an overnight culture * Address correspondence to: George Y. Wu, M.D., Ph.D., Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Rm. AM-044, 263 Farmington Ave., Farmington, CT 06030-1845; tel (860) 679-3158; fax (860) 679-3159; e-mail
[email protected]. 1 Abbreviations: HBV, hepatitis B virus; NEM, N-ethylmaleimide; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid); IPTG, isopropylthio-β-D-galactosidase; SDS-PAGE, sodium deodecyl sulfate-polyacrylamide gel electrophoresis.
was diluted in LB plus 100 µg/mL ampicillin and grown until the OD550 reached 1.0. Isopropylthio-β-D-galactosidase (IPTG) (Gibco, Grand Island, NY) was added to a final concentration of 1 mM, and the culture was grown for an additional 5 h. Cells were harvested by centrifugation at 1500g, 15 min at 4 °C. Cell pellets were resuspended in 0.2 M KCl, 50 mM Tris HCl, pH 7.9, 5 mM MgCl2, 0.1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride and disrupted by sonication on ice using 5 × 15 s bursts at amplitude 40. Cell lysates were clarified by centrifugation, diluted with 50 mM Tris HCl, pH 7.9, 0.1 mM EDTA to an ionic strength equivalent to 50 mM NaCl, 50 mM Tris HCl, pH 7.9, 0.1 mM EDTA. Clarified cell lysates were loaded onto a DEAE-Sepharose column, 5 × 6 cm (Amersham Pharmacia Biotech, Uppsala, Sweden), equilibrated with 50 mM Tris HCl, pH 7.9, 0.15 M NaCl, 0.1 mM EDTA. A DEAE-sepharose column was connected in series to a P-11 phosphocellulose column, 2.5 × 20 cm (Whatman International Ltd, Maidstone, England) equilibrated with the same buffer. RNase H was eluted using 50 mM Tris HCl, pH 7.9, 0.1 mM EDTA, 0.75 M NaCl (1). Aliquots of eluted fractions were run on a 15% SDS-PAGE. Fractions found to contain RNase H were pooled and desalted using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and concentrated through P10 centricon concentrators (Amersham Pharmacia Biotech, Uppsala, Sweden). A Bradford assay was performed to determine protein concentration (12). Conjugation of RNase H and Oligonucleotide. A modified 13-base oligonucleotide (GCAGAGGTGAAGC) specific for the DR1 site on HBV adw strain (13) was synthesized by Keck Oligonucleotide Synthesis Facility at Yale University. The 5′-guanosine was amino-modified at the 6-carbon to provide a substrate for the N-(γmaleimidobutyryloxy) sulfosuccinimide ester, sulfo-GMBS (Pierce Chemical Co, Rockford, IL), linkage, and all other
10.1021/bc010018e CCC: $20.00 © 2001 American Chemical Society Published on Web 09/05/2001
RNase H-Oligodeoxynucleotide Conjugate
residues were phosphorothioate modified. The oligonucleotide, 360 nM, was incubated with a 50-fold molar excess of sulfo-GMBS in PBS pH 7.4 for 1 h at 25 °C. RNase H, 90 nM, was incubated with 100 mM dithiothreitol (DTT) (Sigma, St. Louis, MO) in PBS pH 7.4 at 25 °C for 1 h. Both were purified by elution through a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) with PBS pH 7.4. The peaks from the DR1 oligonucleotideGMBS and the reduced RNase H were pooled and incubated overnight at 4 °C. Conjugate was purified by passing through a HiTrap Q column (Amersham Pharmacia Biotech, Uppsala, Sweden) using a stepwise NaCl gradient, 0.3 M to 1.0 M NaCl (1). Concentrations were determined by Bradford assay and spectrophotometer readings at A260. Conjugation and purity were checked by SDS-PAGE, agarose gels, and radiolabeling experiments. Activity was determined by cleavage assays as described below. SDS-PAGE and Agarose Gels. To visualize the protein and determine the efficacy of purification, 5 µg of RNase H and conjugate (containing the equivalent to 5 µg RNase H) were run on a 10% SDS-PAGE. Bands were visualized by Coomassie Blue staining. To detect DNA in samples of conjugate, 5 µg of free oligonucleotide and conjugate (containing the equivalent of 5 µg of oligo DNA) were run on an 1% agarose gel in 0.045 M Trisborate, 1 mM EDTA buffer, and stained with ethidium bromide to visualize DNA. Radiolabeling. To follow conjugation products and extent of purification, the 3′-OH ends of both oligonucleotides alone and conjugates were labeled with γ-32P ATP using terminal deoxynucleotidyl transferase, Promega, Madison, WI (14). Samples with a specific activity of 2000 cpm/ng were run on a denaturing 12% polyacrylamide gel containing 8 M urea and autoradiographed (15). Cleavage Assays. HBV and control β-globin RNA templates were synthesized from plasmid DNA using T7 polymerase (Gibco Life Technologies, Grand Island, NY). The HBV RNA template was created from an adw R9 plasmid (gift from Dr. T. J. Liang, NIH) resulting in a fragment of 1200 bases, which included the DR1 site. A control transcript consisted of a similar size fragment from nonrelated pLMR3 rabbit β-globin plasmid DNA (gift from Dr. Gordon Carmichael, University of Connecticut). One microgram (0. 25 pmol) of RNA transcript was incubated with 2.5 µmol of RNase H alone, oligonucleotide alone, or a mixture of 2.5 µmol unconjugated RNase H plus 2.5 µmol oligonucleotide, or 5 µmol of conjugate in reaction buffer consisting of 40 mM Tris pH 7.5, 4 mM MgCl2, 1 mM DTT, 150 mM NaCl, plus anti-RNase (Ambion, Austin, TX), which does not inhibit RNase H (Ambion Manufacturer’s note). To assess the effects of inhibitory agents on cleavage activity of the conjugate, 0.25 pmol of HBV transcript was incubated with 5 µmol of oligo-RNase H conjugate alone or plus 25 µM EDTA (ethylenediaminetetraacetic acid), 25 µM EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) or 25 µM NEM (Nethylmaleimide). To determine concentration dependence, cleavage assays were performed using increasing amounts of conjugate with constant amounts of substrate. A comparison of conjugated versus unconjugated RNase H activity was made using 5 µmol of conjugate versus decreasing amounts of unconjugated RNase H and oligonucleotide in equimolar ratios. As a control for nonspecific cleavage, conjugate was added to a nonrelated β-globin RNA transcript. All cleavage assays were performed by incubation for 1 h at 37 °C and run on 2.2 M formaldehyde, 1% agarose gels in MOPS buffer (0.02 M MOPS
Bioconjugate Chem., Vol. 12, No. 5, 2001 771
Figure 1. 10% SDS-PAGE of purified mutant RNase H and oligo-RNase H conjugate prepared as described in the Materials and Methods section. Lane 1, molecular weight marker; lane 2, 5 µg of mutant RNase H; and lane 3, 5 µg of oligo-RNase H conjugate.
Figure 2. An autoradiogram of an RNase H conjugate composed of a 32[P]-oligo linked to mutant RNase H and analyzed on a 12% polyacrymide gel in 8 M urea as described in the Materials and Methods section. Lane 1, 7.5 ng of 32[P]- oligo alone; lane 2, 7.5 ng of 32[P]-oligo-RNase H conjugate. Both samples had a specific activity of 2000 cpm/ng.
[3-(N-morpholino)propanesulfonic acid (Sigma, St. Louis, MO), 8 mM sodium acetate, 1 mM EDTA]. RNA bands were visualized by ethidium bromide staining and quantitated by densitometry (16). RESULTS
From 3 L of LB culture medium, a yield of 32 mg of RNase H was obtained. SDS-PAGE analysis of purified protein revealed a band of approximately 18000 Da, which corresponds to the published size of mutant RNase H (1), Figure 1, lane 2. The oligonucleotide DNA-RNase H conjugate migrated at a position corresponding to approximately 29000 Da in the SDS-PAGE gel, lane 3. To visualize the oligonucleotide DNA component of the RNase H conjugate, and to determine whether the sample had any contaminating free DNA, the conjugate was radiolabeled, electrophoresed, and autoradiographed. Figure 2 shows that the conjugate, lane 2, produced a labeled band migrating much slower than free oligonucleotide, lane 1, as similarly seen on the SDS-PAGE in Figure 1. Contaminating free oligonucleotide was not
772 Bioconjugate Chem., Vol. 12, No. 5, 2001
Figure 3. Panel A. Formaldehyde-agarose gel of cleavage of 0.25 pmol of 1200 bp HBV transcript stained with ethidium bromide. Lane 1, RNA molecular weight markers; lane 2, transcript alone; lane 3, transcript plus 2.5 µmol of RNase H; lane 4, transcript plus 2.5 µmol of oligo; lane 5, transcript plus 2.5 µmol of free oligo plus 2.5 µmol of RNase H; lane 6, transcript plus 5 µmol of oligo-RNase H conjugate; and lane 7, 0.25 pmol of β-globin transcript plus 5 µmol of oligo-RNase H conjugate. Panel B. A graph of densitometric analyses of residual substrate HBV RNA present in the corresponding lanes of which Panel A shows a representative. All assays were performed in triplicate and the results expressed as means ( SD in arbitrary densitometric units.
seen in the conjugate sample, indicating that within the limits of detectability, the purification process successfully eliminated uncoupled oligo DNA. From the protein and DNA assays of the conjugate, it was calculated that the RNase H and oligonucleotide were linked in molar ratio of one to one. To determine whether the conjugated oligonucleotide DNA-RNase H conjugate was functional, cleavage activity was studied using HBV mRNA substrates containing the target DRI sequence. In Figure 3, lane 2, the HBV substrate transcript is shown without exposure to any enzyme. Lane 3 shows that exposure of the HBV substrate to RNase H alone failed to result in any detectable cleavage. Similarly, lane 4 shows that incubation of HBV substrate with oligonucleotide DNA alone had no effect. However, lane 6 shows that transcripts incubated with conjugate were nearly completely digested with little intact substrate remaining. This result is similar to the positive control containing unconjugated RNase H and oligonucleotide mixed in the same proportions as provided by the conjugate and incubated under identical conditions, lane 5. A control, unrelated transcript having no HBV sequences, incubated with conjugate in the same proportions as in lane 6, showed no detectable cleavage, lane 7. Densitometry revealed that incubation of conjugated oligonucleotide DNA-RNase H or the mixture of enzyme plus free oligonucleotide DNA decreased substrate by more than 85%. As controls for nonspecific degradation, digestions of substrate HBV mRNA with oligonucleotide DNA-RNase H conjugate were carried out in the presence of inhibitors. Figure 4 shows that addition of 25 µM EDTA, a magnesium chelator, to the incubation mixture containing RNA
Walton et al.
Figure 4. Panel A. Formaldehyde-agarose gel of 0.25 pmol of HBV transcript cleavage products in the presence of chelating and alkylating agents, and stained with ethidium bromide. Lane 1, RNA molecular weight markers; lane 2, transcript alone; lane 3, transcript plus 5 µmol of oligo-RNase H conjugate; lane 4, same as lane 3 plus 25 µM EDTA; lane 5, same as lane 3 plus 25 µM EGTA; and lane 6, same as lane 3 plus 25 µM NEM. Panel B. A graph of densitometric analyses of residual substrate HBV RNA present in the corresponding lanes of gels which Panel A shows a representative. All assays were performed in triplicate and the results expressed as means ( S. D. in arbitrary densitometric units.
substrate prevented cleavage of HBV transcript by the conjugate, lane 4. In contrast, addition of EGTA, a calcium chelator, lane 5, or NEM, which interferes with cysteine residues, lane 6, had no significant inhibition of cleavage activity. These results are consistent with the known requirement of the RNase H enzyme for magnesium, but not calcium for cleavage activity (1,3,17). The mutant RNase H1 used in these conjugation experiments is known not to be susceptible to inhibition by sulfhydryl regents and, therefore, is not affected by NEM (18). However, it has been shown that NEM does inhibit native RNase H1 by the steric effects of sulfhydryl modification (7). In the current studies, RNase H1 was mutated to eliminate all but one cysteine and that remaining cysteine was modified by linkage to the oligonucleotide DNA, therefore eliminating the availability of cysteine residues for interference by NEM. Furthermore, the data argue against significant contamination with RNase H2 or other RNases susceptible to inhibition by sulfhydryl reactive agents. The dependence of cleavage activity on conjugate concentration is shown in Figure 5. The gel shows that with a constant amount of starting substrate RNA of 0.25 pmol, decreasing the amounts of oligonucleotide-RNase H conjugate from 5 to 0.625 µmol resulted in decreasing amounts of substrate cleaved, lanes 2-6, respectively. Quantitatively, this represented a decrease from 68% at 5 µmol to 24% at 0.625 µmol as shown in panel B. Additional bands can be seen in this gel and correspond with the predicted sizes resulting when the HBV tran-
RNase H-Oligodeoxynucleotide Conjugate
Figure 5. Cleavage of 0.25 pmol of HBV transcript by varying amounts of conjugates assayed by agarose gels stained with ethidium bromide. Panel A. Lane 1, RNA molecular weight markers; lane 2, transcript alone; lane 3, transcript plus 5 µmol of oligo-RNase H conjugate; lane 4, transcript plus 2.5 µmol of conjugate; lane 5, transcript plus 1.25 µmol of conjugate; and lane 6, transcript plus 0.625 µmol of conjugate. Panel B: A graph of densitometric analyses of residual substrate HBV RNA present in the corresponding lanes of gels which Panel A shows a representative. All assays were performed in triplicate and the results expressed as means ( SD in arbitrary densitometric units.
script is cleaved, 180, 230, and 772 nucleotides. In most studies these bands were not seen, most likely due to degradation of the smaller more unstable fragments. To estimate the activity of the conjugated RNase H relative to the unconjugated enzyme, HBV transcript, 0.25 pmol, was incubated with conjugate, 5 µmol, or with varying amounts of free enzyme plus free oligo in equimolar amounts relative to each other. Figure 6 shows that the cleavage activity of 5 µmol of conjugated enzyme, lane 3, was equal to the activity seen with between 0.25 µmol, lane 5, and 0.025 µmol, lane 4, of mixed enzyme and free oligonucleotide. By this calculation, the conjugate was approximately 5% as active as a mixture of free enzyme and oligo on an equimolar basis. DISCUSSION
Although a DNA virus, HBV undergoes replication through a pregenomic RNA intermediate that serves not only as a template for replication, but also as a messenger RNA species (19). Because of its importance in the viral life cycle, cleavage of this intermediate could prevent synthesis of new virus. An agent that could result in sequence specific destruction of this entity could be valuable not only in the study of the HBV replication, but also, potentially as a therapeutic agent. The data presented in this report suggest that chemical linkage of the oligonucleotide to RNase H can confer HBV sequence specificity and still maintain enzymatic activity. Because the enzyme and oligonucleotide DNA are chemically linked, the two components are brought into physical proximity, and therefore have the potential to be
Bioconjugate Chem., Vol. 12, No. 5, 2001 773
Figure 6. Cleavage of 0.25 pmol of HBV transcript with varying concentrations of free oligo and mutant RNase H, assayed by agarose gels stained with ethidium bromide. Panel A. Lane 1, RNA molecular weight markers; lane 2, transcript alone; lane 3, transcript plus 5 µmol of oligo-RNase H conjugate; lane 4, transcript plus 2.5 µmol of free oligo and 2.5 µmol of RNase H; lane 5, transcript plus 0.25 µmol of free oligo and 0.25 µmol of RNase H; lane 6, transcript plus 0.025 µmol of free oligo and 0.025 µmol of RNase H; and lane 7, transcript plus 2.5 nmol of free oligo and 2.5 nmol of RNase H. Panel B: A graph of densitometric analyses of residual substrate HBV RNA present in the corresponding lanes of gels which Panel A shows a representative. All assays were performed in triplicate and the results expressed as means ( SD in arbitrary densitometric units.
delivered together into intracellular compartments. In addition, the chemical linkages created between oligonucleotide-cross-linker-RNase H are noncleavable (20) and should remain stable within intracellular compartments. E. coli and human RNase H1 share many of the same enzymatic properties including the requirement of divalent cations. The sites of cation binding, substrate binding, and enzyme activity are highly conserved in all RNase H molecules (4). Several lysine groups in the molecule’s substrate binding site are thought to interact with the phosphate groups on the substrate, and the site of this interaction is thought to be in the minor groove (1, 3). The cysteine 13 residue was chosen for disulfide linkage of antisense DNA as it is close to the active site on the enzyme, but is not essential for cleavage activity (1). The other cysteine residues, also not necessary for cleavage activity, were mutated to serine and alanine in order not to interfere with sulfo-GMBS linkage of the oligonucleotide. A restriction site was added for cloning purposes to create the mutant RNase H. This molecule was shown to have 90% activity of wild type (1). Ma et al. showed that these modifications do not interfere with binding to the target mRNA. The antisense oligonucleotide used in current system was modified in two ways. The first was the formation of phosphorothioate linkages to inhibit degradation by cellular nucleases. Heteroduplexes consisting of RNA and
774 Bioconjugate Chem., Vol. 12, No. 5, 2001
phosphorothioate-linked DNA have been shown to be suitable substrates for RNase H activity (3, 21). The second was an addition of an amino group to the 5′ carbon. This provided a functional group for modification by a coupling agent for conjugation of the oligonucleotide to the mutated RNase H without interfering with the sequence specificity and binding activities. The mutations in RNase H eliminated other potentially reactive cysteines, thus permitting specific modification and coupling of the oligonucleotide to the cysteine 13 residue in the enzyme. The length of the antisense oligonucleotide DNA is also an important consideration. The minimum RNA-DNA hybrid that has been found to result in cleavage is six nucleotides. Sequences greater than six but less than nine have been still be shown be cleaved but at slower rates (3). However, the longer the length of an oligonucleotide the more likely the possibility of nonspecific binding and cleavage (2). On the basis of this information, a 13-mer sequence was selected to be used in the current studies. The cleavage of the HBV transcript by the conjugated ribonuclease H was expected to result in fragments of 180, 230, and 772 nucleotides, based on the complementary sites predicted to be recognized by the oligonucleotide. Some of the smaller fragments were barely or not detectable in some of the cleavage assays in this current study. It is likely that the resulting cleavage fragments were unstable, and were degraded quickly as has been reported in other studies on antisense oligonucleotides against other target RNAs (22). Successful cleavage was seen when the conjugate was incubated with the HBV RNA, but not with the rabbit β-globin transcript, indicating that the observed cleavage was not due to nonspecific degradation of the target. The fact that neither RNase H nor oligonucleotide alone resulted in cleavage activity confirmed that neither had intrinsic nonspecific nuclease activity or were contaminated with another RNase, and that the presence of both RNase H and DNA together with the RNA substrate was required for cleavage activity. The RNase H activity in the conjugate was found to be less than that of RNase H alone, estimated to be approximately 5% of unconjugated RNase H. This is similar to but higher than activity reported by Ma et al. for a conjugated RNase H against mouse β-globin message in which activity of the conjugate was only 0.3% that of free enzyme mixed with oligonucleotide DNA (1). The difference in results may be due to the differing affinities of the oligonucleotide DNA of their respective target RNA sequences. Experiments combining the catalytic properties of RNase H and the binding specificity of antisense DNA have been used previously to obtain sequence-specific cleavage of the target template (1, 9-11). Most of these studies involved mixing of antisense DNA with the RNase H to obtain specificity of cleavage. However, to be of practical value, both components need to be delivered to the same compartment in effective concentrations. The data in the current studies indicate that it is possible to specifically couple RNase H in an equimolar ratio of enzyme to oligonucleotide, virtually ensuring simultaneous codelivery of both components at that fixed molar ratio, and represents the first demonstration that such a chimeric molecule can specifically cleave an HBV target mRNA. To be useful, delivery of RNase H-antisense oligonucleotide into cells must occur. This could be achieved using lipofectamine, a polycationic lipid, which has been suc-
Walton et al.
cessful in delivering nucleic acids (23), and proteins into cultured cells without loss of biological activity (24). Alternatively, for specific delivery into liver cells, liverspecific carrier consisting of liver cell recognition component and nucleic acid binding component may be employed to bring RNase H-antisense oligonucleotide into liver cells (23). ACKNOWLEDGMENT
The technical assistance and discussions of Ms. Christy Schilling, and secretarial help of Mrs. Rosemary Pavlick, are gratefully acknowledged. This work was supported in part by grants from the NIDDK: DK-42182 (G.Y.W.), and the Herman Lopata Chair in Hepatitis Research (G.Y.W.). LITERATURE CITED (1) Ma, W. P., Hamilton, S. E., Stonwell, J. G., Byrn, S. R., and Davisson, V. J. (1994) Sequence specific cleavage of messenger RNA by a modified ribonuclease H. Bioorg. Med. Chem. 2 (3), 169-179. (2) Eder, P. S., and Walder, J. A. (1991) Ribonuclease H from K562 human erythroleukemia cells. Purification, characterization, and substrate specificity. J. Biol. Chem. 266, 64726479. (3) Frank, P., Albert, S., Cazenave, D., and Toulme J. J. (1994) Purification and characterization of human ribonuclease HII. Nucleic Acid Res. 22, 5247-5254. (4) Nakamura, H., Oda, Y., Iwai, S., Inouse, H., Ohtsuka, E., Kanaya, S., Kimura, S., Katsuda, C., Katayanagi, K., Morikawa, K., Miyashiro, H., and Ikehara, M. (1991) How does RNase H recognize a DNA:RNA hybrid? Proc. Natl..Acad. Sci. U.S.A. 88, 11535-11539. (5) Kanaya, S. (1998) Ribonuclease H. (J. J. Crouch, Ed.) pp 39-66, INSERM, Paris, France. (6) Crouch, R. J., and Dirksen, M. L. (1982) Ribonuclease H. Cold Spring Harbor Monogr. Ser. 14, 211-254. (7) Wu, H., Lima, W. F., and Crooke, S. T. (1999) Properties of cloned and expressed human RNase H. J Biol. Chem. 274 (40), 28270-28278. (8) Manoharan, M. (1999) 2′-Carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation. Biochim. Biophys. Acta 1489, 117-130. (9) Baertschi, A. J. (1994) Antisense oligonucleotide strategies in physiology. Mol. Cell. Endocrinol. 101 (1-2), R 15-24. (10) Veal, G. J., Agrawal, S., and Byrn, R. A. (1998) Sequencespecific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro. Nucleic Acids Res. 26 (24), 5670-5675. (11) Alt, M., Eisenhardt, S., Serwe, M., Renz, R., Engels, J. W., and Caselmann, W. H. (1999) Comparative inhibitory potential of differently modified antisense oligodeoxynucleotides on hepatitis C virus translation. Eur. J. Clin. Invest. 10, 86876. (12) Bradford, M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. (13) McLachlan, A., Ed. (1991) Molecular Biology of the Hepatitis B Virus, pp 20-21, CRC Press Inc., Boca Raton, FL. (14) Tu, C. P., and Cohen, S. N. (1980) 3′-End labeling of DNA with 32[P] cordycepin-5'triphosphate. Gene 10, 177-183. (15) Sambrook, J., Fritsch, E. F., and Maniatis, T., Eds. Gel electrophoresis of DNA, in Molecular Cloning - A laboratory manual., 2nd ed. (1989) p 6.4.5, Cold Spring Harbor Laboratory Press, Plainview, NY. (16) Wienand, U., Schwarz, Z., and Felix, G. (1978) Applications for analysis of mRNAs from maize endosperm. FEBS Lett. 98, 319-323. (17) Blain, S. W., and Goff, S. P. (1996) Differential effects of Moloney murine leukemia virus reverse transcriptase mutations on RNase H activity in Mg2+ and Mn2+. J. Biol. Chem. 271, 1448-1454.
RNase H-Oligodeoxynucleotide Conjugate (18) Kanaya, S., Kimura, S., Katsuda, C., and Ikehara, M. (1990) Role of cysteine residues in ribonuclease H from Escherichia coli. Biochem. J. 271, 59-66. (19) Lau, J. Y. N., and Wright, T. L. (1993) Molecular virology and pathogenesis of hepatitis B. Lancet 342, 1335-1336. (20) Fujiwara, K., Matsumoto, N., Yagisawa, S., Tanimori, H., Kitagawa, T., Hirota, M., Hiratani, K., Fukushima, K., Tomonaga, A., Hara, K., and Yamamoto, K. (1988) Sandwich enzyme immunoassay of tumor-associated antigen sialosylated Lewis x using β-D-galactosidase coupled to a monoclonal antibody of IgM isotype. J. Immunol. Methods 112, 77-83. (21) Dagle, J. M., and Weeks, D. L. (1999) Selective degradation of targeted mRNAs using partially modified oligonucleotides. Methods Enzymol. 313, 420-436.
Bioconjugate Chem., Vol. 12, No. 5, 2001 775 (22) Qui, G., Goodchild, J., Humphreys, R. E., and Xu, M. (1999) Cancer immunotherapy by antisense suppression of Ii protein in MHC-class-II-positive tumor cells. Cancer Immunol. Immunother. 48 (9), 499-506. (23) Wu, C. H., and Wu, G. Y. (1998) Targeted inhibition of Hepatitis C virus -directed gene expression in human hepatoma cell lines. Gastroenterology 114, 1304-1312. (24) LaMartina, S., Roscilli, G., Rinaudo, D., Delmastro, P., and Toniatte, C. (1998) Lipofection of purified adeno-associated virus Rep68 protein: toward a chromosome-targeting nonviral particle. J. Virol. 72, 7653-7658.
BC010018E
