Bioconjugate Chem. 2003, 14, 295−301
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Conjugation of an Antisense Oligodeoxynucleotide to Ribonuclease H Results in Sequence-Specific Cleavage and Intracellular Inhibition of HCV Gene Expression Toshiko Fukuma, Cherie M. Walton, Catherine H. Wu, and George Y. Wu* Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington, Connecticut. Received October 24, 2002; Revised Manuscript Received December 12, 2002
A recombinant E. coli ribonuclease H (RNase H) was chemically coupled to an antisense oligodeoxynucleotide (ODN) against the 5′-noncoding region (5′-NCR) of the hepatitis C virus. Purity of the conjugates was confirmed by sodium deodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a band corresponding to approximately 23 kDa. Conjugate function was tested by the cleavage of a HCV RNA transcript including the 5′-NCR and core region and showed HCV sequence-specific cleavage by the appearance of an expected ∼1000 nt fragment of RNA. Cleavage was not seen by RNase H alone, or ODN alone. Delivery studies using 32P- and 125I-labeling showed that while RNAse H failed to enter cells, the conjugate was efficiently taken into the cells. To assess intracellular effects, a cell line, Huh-7/CMV-NCRC∆luc, which expresses HCV mRNA (nt 1-585) fused to a marker gene, was transfected with the conjugate. Reporter gene expression was suppressed by 51.2% with the conjugate compared to only 39.7% by ODN alone, 35.8% by a mixture of RNase H plus ODN, and not at all by RNase H alone. In conclusion, the RNase H-ODN conjugate effectively cleaved an HCV transcript in vitro and inhibited expression of an HCV-marker fusion construct in a liver-derived cell line.
INTRODUCTION
MATERIALS AND METHODS
Ribonuclease H (RNase H) is an endonuclease that cleaves only the RNA strand of RNA-DNA hybrids (1). However, it does not have sequence-specific properties. It has been isolated from Escherichia coli (2, 3) as well as human cells (4, 5). E. coli RNase H1 is composed of a single polypeptide chain with 155 amino acids, and its properties are well characterized (6-8). Antisense oligodeoxynucleotides (ODNs) can hybridize with target RNA molecules in a sequence-specific manner, blocking translation and resulting in reduction of protein synthesis including that of several viruses (914). Antisense ODNs against the 5′-noncoding region (5′NCR) and core region of hepatitis C virus (HCV) genome were used to inhibit the HCV-directed expression in vitro (15, 16) in human hepatoma cell lines (17-19) and in an animal model infected with an HCV-vaccinia virus recombinant (20). Although endogenous RNase H is suggested to have a role in inhibition of gene expression by ODNs (21-24), the normal RNase H expression level in mammalian cells is low. To obtain high, intracellular concentrations of RNase H and to colocalize it with ODNs, we sought to link RNase H to ODN to form a conjugate. The aim was to synthesize an RNase H-ODN conjugate targeting HCV, test sequence-specific cleavage using HCV RNA transcript in vitro, and study its intracellular activity in a human liver cell line that expresses HCV mRNA.
Purification of RNase H. E. coli strain MIC1066 expressing a mutant RNase H (C63S, C133A) was a generous gift from Dr. Robert Crouch, NIH. RNase H was purified as previously described (25, 26). Antisense ODN Design. A 14-mer HCV 5′-NCR sequence specific antisense ODN was designed against a target sequence based on the predicted secondary structure of the HCV genome, nt 325-338 (5′-CUCGUAGACCGUGC-3′). A 12-base oligonucleotide (5′-GCAGGGTGAAGC-3′) specific for the DR1 site on HBV adw strain was prepared as a negative oligonucleotide control. The ODN was 5′-amino modified, and the backbone phosphorothioate was modified (HHMI/Keck Oligonucleotide Synthesis Facility, Yale University). RNase H-ODN Conjugate Synthesis. RNase HODN conjugate was synthesized as previously described (25, 26). Briefly, 360 nM ODN and 50-fold molar excess water-soluble cross-linker, Sulfo-GMBS. RNase H-ODN conjugates were purified by anion exchange column, HiTrap Q (Amersham Pharmacia Biotech Inc., Piscataway, NJ). SDS-PAGE analysis was performed, and bands were visualized by Coomassie Brilliant Blue. 32 P-Labeling of ODN and 125I-Labeling of RNase H. DNA-labeling of RNase H-ODN conjugate and ODN were performed by 3′-OH end-labeling with R32P-ATP using terminal deoxynucleotidyl transferase according to manufacturer’s instructions (Promega, Madison, WI). Labeled products were analyzed on SDS-polyacrylamide (15%)/urea (8 M) gels, followed by autoradiography. Protein-labeling of RNase H and RNase H-ODN conjugate were performed with 125I by the chloramine-T method and analyzed by SDS-PAGE (15%) and autoradiography.
* 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, (860) 6793158, (860) 679-3159 (FAX), e-mail
[email protected].
10.1021/bc0256300 CCC: $25.00 © 2003 American Chemical Society Published on Web 01/30/2003
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Plasmids and in Vitro Transcription. To evaluate the cleavage properties of RNase H-ODN conjugate, an in vitro cleavage substrate was prepared. The plasmid, p90/HCVFLlong pU, containing a full length HCV cDNA (Genotype Ia, Dr. Charles M. Rice, Rockefeller University, NY), was digested with restriction enzymes Xba I/BamHI to generate a 1,357 bp fragment. This fragment was subcloned into pUC18, resulting in pUC18/T7-HCV (nt 1-1357). As a control, an HBV RNA template was also created from an adw R9 plasmid (Dr. T. J. Liang, NIH) resulting in a fragment of 1182 nt (26). In vitro transcription was performed using conventional T7 RNA polymerase reaction (Life Technologies, Grand Island, NY). In Vitro Transcript Cleavage Assay. To assess cleavage, 0.5 µM HCV RNA of in vitro transcript and varying concentrations of RNase H-ODN conjugate with ranging 6 nM-0.6 µΜ, was incubated at 37 °C for 1 h in cleavage buffer [40 mM Tris-HCl, pH 7.4, 4 mM MgCl2, 1 mM DTT, 150 mM NaCl, 1 µL anti-RNase (Ambion, Austin, TX)]. To RNase H-ODN conjugate samples, 25 mM N-ethylmaleimide (NEM) was added to prevent the nonspecific cleavage by unconjugated RNase H (27). The effect of the chelating agents EDTA and EGTA were also tested. Appearance of expected cleaved fragments was sought using 0.7% agarose 2.2 M formaldehyde gels. RNA bands were visualized by ethidium bromide staining. Cell Lines and RNase H-ODN Conjugate Transfection. A cell line, Huh-7/CMV-NCRC∆luc was used to test the inhibition of HCV mRNA. This cell line was established by transfecting the plasmid pRc/CMVNCRC∆luc that contains HCV cDNA nt 1-585 upstream of a firefly luciferase gene under the control of CMV promoter (28). Huh-7/CMV-luc, which expresses luciferase gene driven by a CMV promoter, was used as a control. These cell lines were cultured in DMEM supplemented with 10% FBS and 250 µg/mL G418. HepG2 derived cells, HepG2 2.2.15, that produce unrelated HBV viral proteins, were also used as controls (29). Transfections of ODN, RNase H, and RNase H-ODN conjugate into the cells were performed by a polycationic lipid method. Cells (2 × 105) were seeded on 24-well tissue culture dishes and incubated for 24 h prior to the transfection. Various amounts of RNase H-ODN conjugates ranging from 0 µg to 1.6 µg were mixed with LipofectAMINE (DOSPA plus DOPE, Invitrogen, Carlsbad, CA) in OPTI-MEM I (Life Technologies). Final protein concentrations ranged from 0 µM to 0.3 µM. Control samples of ODN alone, RNase H alone, and a mixture of RNase H and ODN were also prepared in the identical molar ratios as present in RNase H-ODN conjugate. Cells were incubated at 37 °C with 5% CO2. At 12 h after transfection, culture medium was replaced. Cells were harvested at 3, 6, 12, 24, and 48 h after transfection, and luciferase assays were performed. Delivery Studies. 32P-labeled ODN and 125I-labeled RNase H were used to assess delivery of the conjugate, ODN and RNase H in Huh-7/CMV-NCRC∆luc cells. Transfections were performed at 0.15 µM using radiolabeled materials as described above. Cells were harvested at 3, 6, 12, 24, and 48 h after transfection. Tissue culture medium was collected and cell layers were washed three times with ice-cold PBS, stripping solution (PBS, pH 5, 10 mM EDTA) followed by lysis buffer. All solutions, and cell lysates were collected. 32P-radioactivity was scintillation counted and 125I-radioactivity was measured with gamma counter. Background cell-associated radioactivity was measured by binding assays incubating cells with radiolabeled materials at 4 °C for 2 h. The delivery was
Fukuma et al.
Figure 1. SDS-PAGE analysis of the purified RNase H and RNase H-ODN (HCV) conjugate. Purified RNase H and RNase H-ODN (3 µg) were separated by 15% SDS-PAGE and stained with Coomassie Brilliant Blue. Lane 1, RNase H; lane 2, RNase H-ODN (HCV) conjugate. Migration of the protein size markers is shown on the left.
assessed by intracellular accumulation, the sum of uptake and elimination from the cells. It was calculated by subtraction of background from total cell-associated amount. Results were expressed as means ( SD (n ) 6). Reporter Gene Assays. Luciferase assays were performed using a commercial kit (Promega) with a luminometer model, LB9501. Total cell protein was determined by Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Luciferase activity measurements were performed in duplicate, and normalized to the total protein concentration of each sample. Results are expressed as means ( SD of four independent experiments. The HBs antigen was quantified by Abbott ELISA according to manufacturer’s instructions (Abbott Laboratories, Abbott Park, IL). Statistical Analyses. Statistical analyses were performed using Student’s t-test with p values of
