Mol Biotechnol DOI 10.1007/s12033-014-9751-3
RESEARCH
Inhibition of DNA Replication of Human Papillomavirus by Using Zinc Finger–Single-Chain FokI Dimer Hybrid Takashi Mino • Tomoaki Mori • Yasuhiro Aoyama Takashi Sera
•
Ó Springer Science+Business Media New York 2014
Abstract Previously, we reported that an artificial zincfinger protein (AZP)–staphylococcal nuclease (SNase) hybrid (designated AZP–SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP–SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP–SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP–scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP–SNase. Then, by linkermediated PCR analysis, we confirmed that AZP–scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP–scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP–scFokI hybrid is expected
Electronic supplementary material The online version of this article (doi:10.1007/s12033-014-9751-3) contains supplementary material, which is available to authorized users. T. Mino T. Mori Y. Aoyama T. Sera Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan T. Mori T. Sera (&) Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan e-mail: [email protected]
to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects. Keywords Antiviral therapy Human DNA virus Human papillomavirus Hybrid nuclease Artificial zincfinger protein Single-chain FokI dimer DNA cleavage
Introduction Human papillomaviruses are double-stranded DNA viruses that induce benign proliferative squamous epithelial and fibroepithelial lesions (warts and papillomas). A subgroup of HPV classified as ‘‘high-risk’’ viruses, including HPV types 16, 18, 31, 35, 39, 45, 51, 52, 58, and 59, has been found to be associated with the development of cervical cancer [1, 2], the second most common malignancy in women worldwide [3]. About 90 % of such tumors contain high-risk HPVs, among which HPV-16 and -18 are the most prevalent. Although vaccines for HPV-16 and -18 have been developed and inoculated, HPV vaccines raise questions: a series of reports suggest that the vaccinations have caused side effects such as body convulsions, pains in joints, or difficulty in walking (http://sanevax.org/hpv-vac cines-japan-requires-disclosure-of-side-effects, http://www. mhlw.go.jp/file/05-Shingikai-10601000-Daijinkanboukousei kagakuka-Kouseikagakuka/0000033852.pdf). The Japanese Ministry of Health, Labor and Welfare withdrew its recommendation to use HPV vaccines in girls in last June (https://ajw.asahi.com/article/behind_news/social_affairs/ AJ201306150057). Therefore, effective antiviral therapies/treatments working in a mechanism different from vaccines are clearly needed. Previously, we demonstrated that inhibition of the binding of a viral replication protein to its replication origin by using
123
Mol Biotechnol
an artificial zinc-finger protein (AZP; [4]) is an effective tactic to prevent DNA virus infection in plants [5] and then applied the methodology to HPV. We demonstrated in transient replication assays that both gene- and protein-delivered AZPs, which were designed to block binding of the HPV-18 E2 protein to its replication origin, efficiently inhibited HPV18 DNA replication in mammalian cells [6, 7]. Although our AZP technology is effective for both plant and animal DNA viruses, the mechanism of DNA replication of viruses of interest must be unraveled in advance; at least, the DNAbinding site of a viral replication protein must be sequenced. To strengthen AZP technology further, we explored the feasibility of inhibition of HPV-18 DNA replication by using AZP-based artificial endonucleases [8]. One potential advantage of this approach is that the genome sequence information on DNA viruses of interest should be sufficient for application: we do not need any information on mechanisms of viral replication. In that study, we generated a novel artificial endonuclease by fusing an AZP for HPV-18 to a staphylococcal nuclease (designated SNase), which cleaves DNA as a monomer. Using the resulting hybrid nuclease AZP–SNase, we demonstrated that both transfection of a mammalian expression plasmid for the AZP–SNase and cell-permeable AZP–SNase efficiently inhibited HPV-18 DNA replication in transient replication assays [8]. Although AZP–SNase did not exhibit any significant cytotoxicity in the transduced cell-lines even 12 days after transduction [8], one potential drawback is that the SNase cleaves RNA and single-stranded DNA (ssDNA) as well as double-stranded DNA (dsDNA) [9]. In the present study, we replaced the SNase moiety in the hybrid nuclease with the single-chain FokI dimer (designated scFokI: [10]) to cleave only dsDNA. The FokI restriction endonuclease or the catalytic domain does not cleave RNA and ssDNA [11, 12]. We previously developed the scFokI by connecting two catalytic domains of FokI via a (GGGGS)12-flexible peptide linker and demonstrated that a hybrid nuclease composed of an AZP and the scFokI cleaved dsDNA with equal or greater reactivity than the corresponding dimeric zinc-finger nuclease [10]. Under the reaction conditions, no detectable cleavage of tRNA involved in the reaction buffer was observed (see the supplementary information). In the present study, we examined whether the resulting hybrid nuclease AZP–scFokI inhibited HPV-18 DNA replication in mammalian cells.
Materials and Methods Plasmid Constructions The six-finger AZP used in the present study is one (designated AZPHPV-1 in Ref. [6]) that did not efficiently reduce the
123
replication of HPV-18 DNA in our previous transient replication assays, although the AZP sequence-specifically recognizes a 19-bp DNA, 50 -GAAAACGGTCGGGACCGAA30 , with an apparent dissociation constant of 10 pM [6]. DNA encoding scFokI was prepared as described previously [10]. A mammalian expression plasmid for AZP–scFokI, designated pCMV-AZP–scFokI, was prepared by cloning the AZP and scFokI open reading frames stepwise into a modified pcDNA3.1 (Life Technologies, Carlsbad, CA, USA) plasmid. The modified plasmid contains an N-terminal T7 tag, a C-terminal V5 tag, a nuclear localization signal (NLS) from the simian virus 40 large T antigen, and a multicloning site for AZP and scFokI. The three plasmids used for DNA cleavage and transient replication assays (described below), pRL-E1, pRL-E2, and pOri, were prepared as described previously [6].
Transient Replication Assays Transient replication assays were performed essentially as previously described [6]. A total of 8 9 105 cells of the human cell line 293H (Life Technologies) were plated onto a BioCoat poly-D-lysine 12-well plate (Becton–Dickinson, Franklin Lakes, NJ, USA) and maintained in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 0.1 mM nonessential amino acids and 10 % fetal bovine serum (Life Technologies). Three plasmids necessary for transient replication, pRL-E1 (1.5 lg), pRLE2 (0.17 lg), and pOri (0.17 lg), were cotransfected with pCMV-AZP–scFokI (0.17 lg) or a vacant plasmid, pcDNA3.1 (0.17 lg) using Lipofectamine 2000 (Life Technologies) as we performed previously by using an AZP-expression plasmid in place of pCMV-AZP–scFokI [6]. Three days after transfection, low-molecular-weight DNA was isolated by Hirt extraction [13]. The samples were first treated with HindIII to linearize them. To distinguish between replicated and unreplicated DNAs, onehalf of each sample was then treated with an excess of DpnI to remove the unreplicated, methylated input DNA [14]. DpnI resistance has been used to demonstrate DNA replication in studies with mammalian cells, including studies with HPVs [15, 16]. One percent of the remaining half of each linearized sample was used to confirm that equal amounts of the plasmids used for each transient replication assay were introduced into 293H cells. The DNA samples were separated by electrophoresis (in 0.8 % agarose gels with 0.5 9 Tris–borate-EDTA buffer), followed by Southern blot hybridization. A 200-bp digoxigenin (DIG)-labeled probe specific to an ampicillin-resistance gene was prepared from pUC-19 by PCR using DIG-11-dUTP (Roche Molecular Biology, Tokyo, Japan) and the primer set 50 -CGGCATCAGAGCAGAT
Mol Biotechnol
TGTACTGAGAGTGC-30 and 50 -TACCCAACTTAATCGC CTTGCAGCACATCC-30 . Because pRL-E1, pRL-E2, pCMV-AZP–scFokI, and pOri contain the ampicillin-resistance gene as a selection marker, all these plasmids could be detected by using the DIG-labeled probe. DNAs were resolved in a 0.8 % agarose gel and transferred onto a Nytran SuPerCharge membrane by means of a TurboBlotter (Schleicher & Schuell, Dassel, Germany). After hybridizing with the DIG-labeled probe, DNA bands corresponding to the plasmids used for transient replication assays were recorded on X-ray films, using anti-DIG-AP and CDP-Star according to the accompanying protocols (Roche Molecular Biology). DNA band intensities on X-ray films were digitized and quantitated using UN-SCAN-IT (Silk Scientific, Inc., Orem, UT, USA). Average DNA band intensities of replicated pOri were calculated from three independent experiments and normalized with DNA band intensities of input pRL-E1 or pOri.
Ligation-Mediated PCR (LM-PCR) of DNA DoubleStrand Breaks in 293H Cells by AZP–scFokI Each Hirt-extracted DNA sample from the transient replication assays described above was treated with T4 DNA polymerase to blunt staggered DNA lesions at 12 °C for 15 min. After phenol extraction, the blunted DNA was ligated with DNA Ligation Kit Ver.2.1 (Takara, Shiga, Japan) to 100 pmol of a double-stranded blunt-ended linker, which was generated by annealing 50 -GCGG TGACCCGGGAGATCTGAATTC-30 and 50 -GAATTCA GATC-30 [17], in a total volume of 10 ll overnight at 16 °C. The ligated DNA sample (0.5 ll) was amplified by PCR with the primer set of the linker-specific primer 50 -GCGGTGACCCGGGAGATCTGAATTC-30 and biotin-labeled pOri-specific primer 50 -CAGCTGGCACGACA GGTTTCCCGACTGGAA-30 . The PCR conditions were initial denaturation at 94 °C for 3 min and 10 cycles of heating (94 °C, 30 s), annealing (66 °C, 2 min), and elongation (72 °C, 1 min). The PCR product was purified by using a QIAGEN PCR purification kit (Qiagen, Valencia, CA, USA) and further purified using Dynabeads M-270 Streptavidin (Life Technologies) according to the protocol accompanying the reagent. The purified sample was used as the template for a second round of PCR with the primer set of the linker-specific primer and pOri-specific primer. The PCR conditions were initial denaturation at 94 °C for 3 min and 30 cycles of heating (94 °C, 30 s), annealing (66 °C, 2 min), and elongation (72 °C, 1 min). The reaction mixtures were separated on 2 % agarose gel. The gels were photographed under UV irradiation. The experiment was repeated independently three times, and a representative result is shown.
Analysis of AZP–scFokI Cytotoxicity The cytotoxicity of AZP–scFokI was evaluated by a modified MTT (methyl thiazolyl tetrazolium) assay with a highly water-soluble disulfonated tetrazolium salt [18], which is commercially available as a Cell Counting Kit-8 from Dojindo (Kumamoto, Japan). A total of 6 9 104 of 293H cells were plated onto a BioCoatTM poly-D-lysine 96-well plate (Becton–Dickinson) and maintained in 0.1 ml of Dulbecco’s modified Eagle’s medium supplemented with 0.1 mM nonessential amino acids and 10 % fetal bovine serum. pRL-E1 (75 ng), pRL-E2 (8.3 ng), and pOri (8.3 ng) were then cotransfected with pCMV-AZP– scFokI or pcDNA3.1 (8.3 ng) as described in transient replication assays. After incubation for 3 d, 10 ll of the solution from the Cell Counting Kit-8 was added into each well, and the plate was incubated at 37 °C for 2 h. The cytotoxicity of AZP–scFokI was evaluated by measuring the absorbance at 450 nm with an ARVO SX 1420 multilabel counter (Wallac, Freiburg, Germany). The experiments were carried out in triplicate and repeated independently three times. Statistical analysis was performed using two-tailed Student’s t-test using Excel (Microsoft).
Results Design of AZP–scFokI To examine the feasibility of inhibition of HPV-18 replication by our novel zinc-finger nuclease, we used a previously reported six-finger AZP [6] as the DNA-binding domain. The AZP was designed using our nondegenerate recognition code table [4] to prevent E2, a replication protein of HPV-18, from binding to its replication origin, leading to inhibition of HPV-18 DNA replication [6]. Although the AZP had a great affinity toward its binding site (its apparent dissociation constant: 10 pM), the tight binding alone was not enough to inhibit HPV-18 DNA replication [6]. Furthermore, we previously confirmed specific binding of the AZP to the HPV-18 replication origin in living 293H cells because inhibition of HPV-18 DNA replication by AZP–SNase was attenuated with an increasing number of mutations of the AZP-binding site in the HPV-18 ori plasmid [8]. In the present study, the AZP was fused with scFokI as a DNA-cleavage domain to generate AZP–scFokI. The hybrid nuclease AZP–scFokI contains a T7 tag, the AZP, scFokI, an NLS from the SV40 large T antigen, and a V5 tag from the N-terminus to the C-terminus and was placed under the control of the human cytomegalovirus promoter (see Fig. 1a). In addition, a vacant plasmid, pcDNA3.1, was used as a control (Fig. 1a).
123
Mol Biotechnol
Inhibition of HPV-18 DNA Replication by AZP–scFokI
a pCMV-AZP-scFokI
GGGGS T7-tag
PCMV
(GGGGS)3 AZP
V5-tag
scFokI
NLS
pcDNA3.1 PCMV
b
39
69
GTAACC GAAAAC GGTCGGGACCGAA AACGGT
AZP-binding site ori
pOri
HPV-18 ori Xmn I
Ampr
c
1
d
2
1
2
pCMV-AZP-scFokI pcDNA3.1 pRL-E1 pRL-E2
Replicated pOri
pCMV-AZP-scFokI
pcDNA3.1
pCMV-AZP-scFokI
pcDNA3.1
100
RESEARCH
Inhibition of DNA Replication of Human Papillomavirus by Using Zinc Finger–Single-Chain FokI Dimer Hybrid Takashi Mino • Tomoaki Mori • Yasuhiro Aoyama Takashi Sera
•
Ó Springer Science+Business Media New York 2014
Abstract Previously, we reported that an artificial zincfinger protein (AZP)–staphylococcal nuclease (SNase) hybrid (designated AZP–SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP–SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP–SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP–scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP–SNase. Then, by linkermediated PCR analysis, we confirmed that AZP–scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP–scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP–scFokI hybrid is expected
Electronic supplementary material The online version of this article (doi:10.1007/s12033-014-9751-3) contains supplementary material, which is available to authorized users. T. Mino T. Mori Y. Aoyama T. Sera Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan T. Mori T. Sera (&) Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan e-mail: [email protected]
to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects. Keywords Antiviral therapy Human DNA virus Human papillomavirus Hybrid nuclease Artificial zincfinger protein Single-chain FokI dimer DNA cleavage
Introduction Human papillomaviruses are double-stranded DNA viruses that induce benign proliferative squamous epithelial and fibroepithelial lesions (warts and papillomas). A subgroup of HPV classified as ‘‘high-risk’’ viruses, including HPV types 16, 18, 31, 35, 39, 45, 51, 52, 58, and 59, has been found to be associated with the development of cervical cancer [1, 2], the second most common malignancy in women worldwide [3]. About 90 % of such tumors contain high-risk HPVs, among which HPV-16 and -18 are the most prevalent. Although vaccines for HPV-16 and -18 have been developed and inoculated, HPV vaccines raise questions: a series of reports suggest that the vaccinations have caused side effects such as body convulsions, pains in joints, or difficulty in walking (http://sanevax.org/hpv-vac cines-japan-requires-disclosure-of-side-effects, http://www. mhlw.go.jp/file/05-Shingikai-10601000-Daijinkanboukousei kagakuka-Kouseikagakuka/0000033852.pdf). The Japanese Ministry of Health, Labor and Welfare withdrew its recommendation to use HPV vaccines in girls in last June (https://ajw.asahi.com/article/behind_news/social_affairs/ AJ201306150057). Therefore, effective antiviral therapies/treatments working in a mechanism different from vaccines are clearly needed. Previously, we demonstrated that inhibition of the binding of a viral replication protein to its replication origin by using
123
Mol Biotechnol
an artificial zinc-finger protein (AZP; [4]) is an effective tactic to prevent DNA virus infection in plants [5] and then applied the methodology to HPV. We demonstrated in transient replication assays that both gene- and protein-delivered AZPs, which were designed to block binding of the HPV-18 E2 protein to its replication origin, efficiently inhibited HPV18 DNA replication in mammalian cells [6, 7]. Although our AZP technology is effective for both plant and animal DNA viruses, the mechanism of DNA replication of viruses of interest must be unraveled in advance; at least, the DNAbinding site of a viral replication protein must be sequenced. To strengthen AZP technology further, we explored the feasibility of inhibition of HPV-18 DNA replication by using AZP-based artificial endonucleases [8]. One potential advantage of this approach is that the genome sequence information on DNA viruses of interest should be sufficient for application: we do not need any information on mechanisms of viral replication. In that study, we generated a novel artificial endonuclease by fusing an AZP for HPV-18 to a staphylococcal nuclease (designated SNase), which cleaves DNA as a monomer. Using the resulting hybrid nuclease AZP–SNase, we demonstrated that both transfection of a mammalian expression plasmid for the AZP–SNase and cell-permeable AZP–SNase efficiently inhibited HPV-18 DNA replication in transient replication assays [8]. Although AZP–SNase did not exhibit any significant cytotoxicity in the transduced cell-lines even 12 days after transduction [8], one potential drawback is that the SNase cleaves RNA and single-stranded DNA (ssDNA) as well as double-stranded DNA (dsDNA) [9]. In the present study, we replaced the SNase moiety in the hybrid nuclease with the single-chain FokI dimer (designated scFokI: [10]) to cleave only dsDNA. The FokI restriction endonuclease or the catalytic domain does not cleave RNA and ssDNA [11, 12]. We previously developed the scFokI by connecting two catalytic domains of FokI via a (GGGGS)12-flexible peptide linker and demonstrated that a hybrid nuclease composed of an AZP and the scFokI cleaved dsDNA with equal or greater reactivity than the corresponding dimeric zinc-finger nuclease [10]. Under the reaction conditions, no detectable cleavage of tRNA involved in the reaction buffer was observed (see the supplementary information). In the present study, we examined whether the resulting hybrid nuclease AZP–scFokI inhibited HPV-18 DNA replication in mammalian cells.
Materials and Methods Plasmid Constructions The six-finger AZP used in the present study is one (designated AZPHPV-1 in Ref. [6]) that did not efficiently reduce the
123
replication of HPV-18 DNA in our previous transient replication assays, although the AZP sequence-specifically recognizes a 19-bp DNA, 50 -GAAAACGGTCGGGACCGAA30 , with an apparent dissociation constant of 10 pM [6]. DNA encoding scFokI was prepared as described previously [10]. A mammalian expression plasmid for AZP–scFokI, designated pCMV-AZP–scFokI, was prepared by cloning the AZP and scFokI open reading frames stepwise into a modified pcDNA3.1 (Life Technologies, Carlsbad, CA, USA) plasmid. The modified plasmid contains an N-terminal T7 tag, a C-terminal V5 tag, a nuclear localization signal (NLS) from the simian virus 40 large T antigen, and a multicloning site for AZP and scFokI. The three plasmids used for DNA cleavage and transient replication assays (described below), pRL-E1, pRL-E2, and pOri, were prepared as described previously [6].
Transient Replication Assays Transient replication assays were performed essentially as previously described [6]. A total of 8 9 105 cells of the human cell line 293H (Life Technologies) were plated onto a BioCoat poly-D-lysine 12-well plate (Becton–Dickinson, Franklin Lakes, NJ, USA) and maintained in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 0.1 mM nonessential amino acids and 10 % fetal bovine serum (Life Technologies). Three plasmids necessary for transient replication, pRL-E1 (1.5 lg), pRLE2 (0.17 lg), and pOri (0.17 lg), were cotransfected with pCMV-AZP–scFokI (0.17 lg) or a vacant plasmid, pcDNA3.1 (0.17 lg) using Lipofectamine 2000 (Life Technologies) as we performed previously by using an AZP-expression plasmid in place of pCMV-AZP–scFokI [6]. Three days after transfection, low-molecular-weight DNA was isolated by Hirt extraction [13]. The samples were first treated with HindIII to linearize them. To distinguish between replicated and unreplicated DNAs, onehalf of each sample was then treated with an excess of DpnI to remove the unreplicated, methylated input DNA [14]. DpnI resistance has been used to demonstrate DNA replication in studies with mammalian cells, including studies with HPVs [15, 16]. One percent of the remaining half of each linearized sample was used to confirm that equal amounts of the plasmids used for each transient replication assay were introduced into 293H cells. The DNA samples were separated by electrophoresis (in 0.8 % agarose gels with 0.5 9 Tris–borate-EDTA buffer), followed by Southern blot hybridization. A 200-bp digoxigenin (DIG)-labeled probe specific to an ampicillin-resistance gene was prepared from pUC-19 by PCR using DIG-11-dUTP (Roche Molecular Biology, Tokyo, Japan) and the primer set 50 -CGGCATCAGAGCAGAT
Mol Biotechnol
TGTACTGAGAGTGC-30 and 50 -TACCCAACTTAATCGC CTTGCAGCACATCC-30 . Because pRL-E1, pRL-E2, pCMV-AZP–scFokI, and pOri contain the ampicillin-resistance gene as a selection marker, all these plasmids could be detected by using the DIG-labeled probe. DNAs were resolved in a 0.8 % agarose gel and transferred onto a Nytran SuPerCharge membrane by means of a TurboBlotter (Schleicher & Schuell, Dassel, Germany). After hybridizing with the DIG-labeled probe, DNA bands corresponding to the plasmids used for transient replication assays were recorded on X-ray films, using anti-DIG-AP and CDP-Star according to the accompanying protocols (Roche Molecular Biology). DNA band intensities on X-ray films were digitized and quantitated using UN-SCAN-IT (Silk Scientific, Inc., Orem, UT, USA). Average DNA band intensities of replicated pOri were calculated from three independent experiments and normalized with DNA band intensities of input pRL-E1 or pOri.
Ligation-Mediated PCR (LM-PCR) of DNA DoubleStrand Breaks in 293H Cells by AZP–scFokI Each Hirt-extracted DNA sample from the transient replication assays described above was treated with T4 DNA polymerase to blunt staggered DNA lesions at 12 °C for 15 min. After phenol extraction, the blunted DNA was ligated with DNA Ligation Kit Ver.2.1 (Takara, Shiga, Japan) to 100 pmol of a double-stranded blunt-ended linker, which was generated by annealing 50 -GCGG TGACCCGGGAGATCTGAATTC-30 and 50 -GAATTCA GATC-30 [17], in a total volume of 10 ll overnight at 16 °C. The ligated DNA sample (0.5 ll) was amplified by PCR with the primer set of the linker-specific primer 50 -GCGGTGACCCGGGAGATCTGAATTC-30 and biotin-labeled pOri-specific primer 50 -CAGCTGGCACGACA GGTTTCCCGACTGGAA-30 . The PCR conditions were initial denaturation at 94 °C for 3 min and 10 cycles of heating (94 °C, 30 s), annealing (66 °C, 2 min), and elongation (72 °C, 1 min). The PCR product was purified by using a QIAGEN PCR purification kit (Qiagen, Valencia, CA, USA) and further purified using Dynabeads M-270 Streptavidin (Life Technologies) according to the protocol accompanying the reagent. The purified sample was used as the template for a second round of PCR with the primer set of the linker-specific primer and pOri-specific primer. The PCR conditions were initial denaturation at 94 °C for 3 min and 30 cycles of heating (94 °C, 30 s), annealing (66 °C, 2 min), and elongation (72 °C, 1 min). The reaction mixtures were separated on 2 % agarose gel. The gels were photographed under UV irradiation. The experiment was repeated independently three times, and a representative result is shown.
Analysis of AZP–scFokI Cytotoxicity The cytotoxicity of AZP–scFokI was evaluated by a modified MTT (methyl thiazolyl tetrazolium) assay with a highly water-soluble disulfonated tetrazolium salt [18], which is commercially available as a Cell Counting Kit-8 from Dojindo (Kumamoto, Japan). A total of 6 9 104 of 293H cells were plated onto a BioCoatTM poly-D-lysine 96-well plate (Becton–Dickinson) and maintained in 0.1 ml of Dulbecco’s modified Eagle’s medium supplemented with 0.1 mM nonessential amino acids and 10 % fetal bovine serum. pRL-E1 (75 ng), pRL-E2 (8.3 ng), and pOri (8.3 ng) were then cotransfected with pCMV-AZP– scFokI or pcDNA3.1 (8.3 ng) as described in transient replication assays. After incubation for 3 d, 10 ll of the solution from the Cell Counting Kit-8 was added into each well, and the plate was incubated at 37 °C for 2 h. The cytotoxicity of AZP–scFokI was evaluated by measuring the absorbance at 450 nm with an ARVO SX 1420 multilabel counter (Wallac, Freiburg, Germany). The experiments were carried out in triplicate and repeated independently three times. Statistical analysis was performed using two-tailed Student’s t-test using Excel (Microsoft).
Results Design of AZP–scFokI To examine the feasibility of inhibition of HPV-18 replication by our novel zinc-finger nuclease, we used a previously reported six-finger AZP [6] as the DNA-binding domain. The AZP was designed using our nondegenerate recognition code table [4] to prevent E2, a replication protein of HPV-18, from binding to its replication origin, leading to inhibition of HPV-18 DNA replication [6]. Although the AZP had a great affinity toward its binding site (its apparent dissociation constant: 10 pM), the tight binding alone was not enough to inhibit HPV-18 DNA replication [6]. Furthermore, we previously confirmed specific binding of the AZP to the HPV-18 replication origin in living 293H cells because inhibition of HPV-18 DNA replication by AZP–SNase was attenuated with an increasing number of mutations of the AZP-binding site in the HPV-18 ori plasmid [8]. In the present study, the AZP was fused with scFokI as a DNA-cleavage domain to generate AZP–scFokI. The hybrid nuclease AZP–scFokI contains a T7 tag, the AZP, scFokI, an NLS from the SV40 large T antigen, and a V5 tag from the N-terminus to the C-terminus and was placed under the control of the human cytomegalovirus promoter (see Fig. 1a). In addition, a vacant plasmid, pcDNA3.1, was used as a control (Fig. 1a).
123
Mol Biotechnol
Inhibition of HPV-18 DNA Replication by AZP–scFokI
a pCMV-AZP-scFokI
GGGGS T7-tag
PCMV
(GGGGS)3 AZP
V5-tag
scFokI
NLS
pcDNA3.1 PCMV
b
39
69
GTAACC GAAAAC GGTCGGGACCGAA AACGGT
AZP-binding site ori
pOri
HPV-18 ori Xmn I
Ampr
c
1
d
2
1
2
pCMV-AZP-scFokI pcDNA3.1 pRL-E1 pRL-E2
Replicated pOri
pCMV-AZP-scFokI
pcDNA3.1
pCMV-AZP-scFokI
pcDNA3.1
100
