:: Asian Journal of Andrology ::

Preparation and identification of activity of anti-HPV-6b/11E1universal ribozymeRz1198 in vitro

De-Zhong LIU^1,2, You-Xin JIN¹, Hua HOU^1,2, Yang-Zhong HUANG² Guang-Cai YANG², Qian XU²

¹State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai 200031,China
²Department of Biochemistry- First Military Medical University, Guangzhou 510515, China

Asian J Androl 1999 Dec; 1: 195-201

Keywords: ribozyme; HPV-6b/11; genetic transcription; cleavage; identification of activity

Abstract

Aim: To study the preparation and cleavage activity of Rz1198 directed against HPV-6bE1 and HPV-11E1 (HPV-6b/11E1) transcripts in vitro. Methods: HPV-6b/11E1 gene fragments were cloned into T-vector under the control of T7 promoter. ³²P-labeled HPV-6b/11E1 transcripts as target-RNAs were transcribed in vitro and purified by PAGE. Rz1198 gene designed as a universal ribozyme for both HPV-6b/11E1 transcripts was cloned into vector p1.5 between 5-cis-Rz and 3-cis-Rz. ³²P-labeled Rz1198 transcript was gel-purified, incubated with target-RNAs at different conditions and autoradiographed after denaturing gel-electrophoresis. Results: Rz1198 was active at 37. The optimal temperature was 50. For HPV-6bE1, k_m=12.2 nmol/L, k_cat=0.18 min^-1; For HPV-11E1, k_m=14.7 nmol/L, k_cat=0.14 min^-1. All these revealed that the design of Rz1198 was correct. It could be a universal ribozyme for the two substratesHPV-6bE1 and HPV-11E1 transcripts. Conclusion: Rz1198 prepared in vitro possesses the perfect specific catalytic cleavage activity. It leads to the expectation that, in the future, it will be possible to develop a new nucleic acid drug from Rz1198 which can efficiently inhibit the replication of HPV-6b/11 DNA in vivo.

1 Introduction

Condyloma acuminatum (CA), a sex transmitted disease (STD), is a benign hyperproliferative disease of the cutaneous and mucosal epithelia induced mainly by human papillomavirus 6b and/or 11 (HPV-6b/11)^[1]. In recent 30 years, the incidence of CA has been continuously increasing in the whole world, and it becomes one of the most familiar STD in our country too. Up to date, possible therapeutic methods include local drug-therapy, physiotherapy, surgery, anti-sense gene therapy, etc., but the effectiveness of all these methods is low and can not effect a radical cure. The disease is easy to recur and is one of the troublesome virus diseases for the clinicians.

Ribozyme is a kind of catalytic RNAs. Some of them possess enzyme-like RNA cleavage activities. In the last decade, the studies on ribozyme for genetic therapy have been in the ascendant and progressed rapidly^[2]. There have been many reports about successful inhibition of the expression of virus genes or other harmful genes by means of ribozyme. Currently, an anti-HIV ribozyme is being tested in two separate Phase I clinical trials^[3]. It is possible that ribozymes would be genetic therapy agents and, in the future, may play an important role in designing strategies for genetic therapy. Alvarez reported^[4] that cis-expression of the hairpin (HP) ribozyme with HPV-16 E6/E7 genes in normal human keratinocytes reduced the growth rate and prevented immortalization. RNA analysis by RT-PCR showed that E6/E7 transcripts were cleaved in post-transfected cells and virtually were eliminated after long-term expression. It demonstrated that the cleavage activity of ribozyme was very high. Huang had also successfully designed anti-HPV-16 E7 hammerhead ribozyme that possessed a biological catalytic activity and expressed stably in CV-1 cells^[5,6]. However, so far there has been no report about the in vitro and in vivo study of ribozyme against the mRNAs of HPV-6b and/or HPV-11the pathogeny of CA.

The study of HPV genomic DNA has showed^[7] that HPV DNA replication initiated after the 63-kDa early protein E1 coded by 2-kb E1 ORF binds to E1-BS (E1 binding site) within HPV Ori (replication origin). E1 protein likely facilitates DNA unwinding by virtue of its helicase and ATPase activities. As a trans-acting factor, E1 is necessary for HPV DNA replication and possesses the most homology among all types of HPV. We thought that if a ribozyme directed against HPV-6b/11E1 mRNA was utilized to disturb the expression of E1 protein, the replication of HPV DNA would be inhibited correspondingly. Thus the proliferation of HPV-6b&11 in affected parts would be controlled and consequently, the immune mechanism of the organism could eliminate the viruses with resultant recovery from CA. The ribozyme designing based on the homology between HPV-6bE1 and HPV-11E1 is hopeful to be an effective genetic therapy for CA.

Analyzing the homologous sequences between HPV-6b&11E1 mRNA by computer, we have designed a universal ribozyme-Rz1198 that could cleave HPV-6b and 11E1 mRNA simultaneously. It was proved by in vitro experiments that Rz1198 cleaved its target-mRNA specifically.

2 Materials and methods

2.1 Materials

E. coli DH5 has been maintained in our laboratory. The ribozyme vector p1.5 that possesses self-cleavage ribozymes (cis-ribozymes) was kindly presented by Professor Qi GR of this Institute. HPV-6b and HPV-11 genomic DNA plasmids were kindly presented by Dr Guo HY of the Third Military Medical University and Dr Wu XB of the Beijing Virus Institute, respectively. DNA sequencing Kit, in vitro transcription Kit, pGEM-T EASY vector, restriction endonucleases, T₄ DNA ligase, RNase A free DNase I and Taq DNA polymerase were purchased from Promega Company; RNasin from Takara Company, and ۦ-³²PdATP andۦ-³²PUTP from Beijing Yahui Company. Materials used were all of analytical purity.

PCR primers: upper primers: for HPV-6b: 5-GAA TTC GTA TTT AGG TAG TCC ATA TGT-3-(nt10861106), for HPV11: 5-GAA TTC AGC AAT GTA GCT AAT GCA GTA-3(nt11171136). Lower primer is universal for the two: 5-GCT CTA GAC GCC ATG TTT CTC TAC CTG-3 (HPV-6b is nt1294-1276; HPV-11 is nt1288-1270).

Oligonucleotides of Rz1198: R1 5-CTA GAG TCG CTT CTG ATG AGT CCG TGA GGA CGA AAC CTT TTT TGG TAC-3; R2: 5-CAA AAA AGG TTT CGT CCT CAC GGA CTC ATC AGA AGC GAC T-3.

They were chemically synthesized in Beckman Oligo-1000 DNA Synthesizer.

2.2 Methods

2.2.1 Construction of in vitro transcription plamids for target-RNAspTV6bE1 & pTV11E1

PCR primers (see 2.1) were designed according to Rz1198 cleavage site at E1 mRNA for HPV-6b and 11. PCR fragments of HPV-6b/11E1 from the two genomic DNA plasmids were purified on 1 % agorase gel and ligated with pGEM-T EASY vectors. DNA sequencing results showed that HPV-6b/11E1 PCR-amplified fragments were cloned into the multiple cloning site of pGEM-T EASY vectors under the control of T7 promoter. The two reconstructed plasmids were named as pTV6bE1 and pTV11E1.

2.2.2 In vitro transcription and purification of target RNA

In vitro transcription was carried out at 37 for 90 min in a 20 L final volume containing 40 mmol/L of Tris-HCl (pH 7.5), 5 mmol/L of DTT, 2 mmol/L of Spermidine, 8 mmol/L of MgCl₂, 0.25 mmol/L of ATP, GTP, and CTP, and 0.05 mmol/L of UTP, 370 kBq ۦ-³²PUTP, 20 U T7 RNA polymerase and 1 g SalI-linearized template(pTV6bE1/pTV11E1) DNA. Target RNA samples were purified by cutting off the autoradiograph bands after running a 6 % polyacrylamide 8-M urea gel and soaking in NES (0.5 mol/L NH₄Ac, 0.1 mol/L EDTA, 0.1 % SDS pH 5.4) at 37 overnight. The products were precipitated by ethanol, washed once by 70 % ethanol, dissolved in DEPC H₂O and reserved under -20.

2.2.3 Construction of in vitro transcription plasmid for ribozyme

In vector p1.5, there was a 1.5 kb fragment between XbaI site in the downstream of 5-cis-Rz and KpnI site in the upstream of 3-cis-Rz (Figure 1). The 1.5 kb fragment was arranged for the convenient isolation of the vector after restriction enzyme digestion and had no other special structure and function.

The universal hammerhead ribozyme-Rz1198 for HPV-6b/11E1 was designed according to the computer software compiled by Professor Chen NA of this Institute. The homologous possibility with the gene of human beings was excluded by consulting with the RNA sequence of human cell from NCBI Gene bank. The vector p 1.5 was digested with the XbaI and KpnI restriction enzymes to release the 1.5 kb fragment. The lineared vector p1.5 without 1.5 kb fragment was purified by 1% agarose gel electrophoresis. After annealing, the two oligonucleotides (R1 & R2) of Rz1198 were ligated into lineared vector p1.5. The reconstructed plasmid was named pR1198 (Figure 1). Rz1198 gene was between 3-cis-Rz and 5-cis-Rz which was just at the downstream of T7 promoter. The reconstruction was affirmed by DNA sequencing.

Figure 1.The map of pR1198 (showing the structure of Rz1198 plus 5-cis-Rz and 3-cis-Rz)

2.2.4 Preparation and purification of Rz1198

The template pR1198 was linearized with SalI, and in vitro transcription was as 2.2.2. The gel was autoradiographed after the transcripts ran a 15% polyacrylamide 8-M urea gel. There are three bands: 5-cis-Rz(58 nt), 3-cis-Rz(43 nt) and Rz1198 (63 nt) (Figure 2A). The 63 nt band was cut off from the gel and purified Rz1198 was acquired. To get large amounts of the transcribed Rz1198 and for initial reaction (II), transcription without isotope was set up as described above except that the cold UTP used was also 0.25 mmol/L. The DNA templates were digested with 20 U RNase A free DNase I. RNA was precipitated by iso-propyl alcohol, dissolved in DEPC H₂O and measured by spectrophotometer. The RNA acquired contained Rz1198 together with 5-cis-Rz and 3-cis-Rz.

Figure 2. In vitro transcripts. (A) In vitro transcript of pR1198. Rz1198 is 63 nt, 58 nt and 43 nt are 5-cis-Rz and 3-cis-Rz, respectively. (B) In vitro transcripts of pTV6bE1 and pTV11E1. Lane 1 (320 nt) is 6bE1 RNA, lane 2 (283 nt) is 11E1 RNA.

2.2.5 In vitro cleavage reaction of Rz1198

Rz1198 and target RNA were quantified by measuring their radioactive Bq value of 1 L solution. The cleavage reaction was carried out in 5 L solution containing 50 mmol/L Tris-HCl (pH 7.5), 20 mmol/L MgCl₂, and 20 mmol/L NaCl. The substrate in one assay was 3.3-6.7 kBq. The molar ratio between Rz1198 and substrate RNA could be estimated according to the Bq number combined with the U number in their RNAs. The initial experiments were: (I) Rz:S=1:1 (mol/L) ratio, 37, 90 min; (II) the condition was as (I), but used cold Rz1198 (it also contained 5-cis-Rz and 3-cis-Rz ); (III) the condition was as (I), but the substrate was 6bE1+11E1. At a 1:4 Rz1198-to-substrate molar ratio, the cleavage mixtures were incubated at different temperatures and different times, as the complete research. To finish the reaction, 1 L Stop Solution (0.25% Bromophenol Blue, 0.25% Xylene cyanol FF, 20 mmol/L EDTA and saturated Urea) was added. After run a 6% denaturing PAGE and autoradiographed, the cleavage results could be analyzed. The cleavage efficiency (CE) was calculated from the Bq values of the bands of substrates (S) and products (P): CE=P/(S+P)ݡ100 %.

2.2.6 Kinetics of the reaction

The procedure was described by Uhlenbeck^[8]. k_m and k_cat were calculated by the Lineweaver-Burk method (double-reciprocal plot).

3 Results

3.1 Identification of transcription of pTV-6bE1 and pTV-11E1

The lengths of RNA transcribed from SalI-linearized templates should be 320 nt (6bE1) and 283 nt (11E1). The results (Figure 2B) were in consistent with our design and proved the correctness of our reconstruction.

3.2 Identification of cleavage activity of Rz1198 prepared in vitro

The cleavage results demonstrated that the designed universal ribozymeRz1198 had the correct structure; it could cleave HPV-6b/11E1 mRNA exactly and efficiently produce two fragments: 184 nt/136 nt and 153 nt/130 nt. Temperature and time could affect cleavage efficiency.

3.2.1 Results of initial experiments

The cleavage efficiency of initial (I): for HPV-6bE1 was 82.8 %; for HPV-11E1 was 73.4 %. Results of (II) and (III) were similar to (I) (data not shown). The results could tell us that the two cis-ribozymes did not disturb the cleavage activity of Rz1198 and Rz1198 could cleave the two substrates in one reaction. When Rz1198 concentration was higher (Rz:S=1:1), the cleavage efficiency increased obviously and an ideal effect could be exerted at a physiological temperature (37).

3.2.2 Temperature course

When the ratio for hot Rz1198 to target RNA was 1:4 (molar ratio), the reaction mixtures were incubated at different temperatures for 60 min (Figure 3A). The optimal temperature was 50 for the two substrates. Figure 3B showed that the cleavage efficiency increased at higher temperature within 0-50桪a typical catalytic activity. The efficiency at 37 was over 30%, but when the temperature was above 50, the cleavage efficiency decreased, as the combination between ribozyme and target was weakened.

Figure 3. Temperature course. (A) Specific cleavage of HPV-6b/11E1 RNA target molecules by Rz1198 prepared in vitro at different temperatures for 60 min. Lane 1-8 are for 6bE1; 9-16 for 11E1. Lane 1 and 9: substrate controls; lane 2 and 10: incubated at 0; lane 3 and 11: at 25; lane 4 and 12: at 37; lane 5 and 13: at 42; lane 6 and 14: at 50; lane 7 and 15: at 55; lane 8 and 16: at 60. It also showed that target-RNA tended to degradation at high temperature. (B) Temperature curves of cleavage reactions of Rz1198 prepared in vitro. -- is for HPV-6bE1;-- is for HPV-11E1.

3.2.3 Time course

When the reaction mixtures (Rz:S=1:4, mol/L) were incubated at 50 for different times, it was shown that the reaction products increased with the increase in incubation time and within 60 minutes it was linear. Longer incubation could obtain more products (Figure 4 A and B).

Figure 4. Time course. (A) Specific cleavage of HPV-6b/11E1 RNA target molecules by Rz1198 prepared in vitro at 50 (optimal temperature) for different times. Lane 1-8 are for 6bE1; 9-16 for 11E1 Lane 1 and 9: substrate controls; lane 2 and 10: incubate for 3 min; lane 3 and 11: for 6 min; lane 4 and 12: for 10 min; lane 5 and 13: for 20 min; lane 6 and 14: for 40 min; lane 7 and 15: for 90 min; lane 8 and 16: for 120 min. (B) Time curves of cleavage reactions of Rz1198 prepared in vitro. -- is for HPV-6bE1; -- is for HPV-11E1.

3.2.4 Mechanism

Under the condition of 50 and 10-min reaction time, the cleavage efficiency was calculated at Rz: S =1:2, 1:4, 1:8, 1:16 and 1:32 (mol/L) ratios. k_m and k_cat were obtained by the Lineweaver-Burk method (Figure 5). For HPV-6bE1, k_m=12.2 nmol/L, k_cat=0.18 min^-1; for HPV-11E1, 14.7 nmol/L and 0.14 min^-1.

Figure 5. Lineweaver-Burk kinetic plots of the specific cleavage of E1 RNA target molecules by Rz1198 prepared in vitro. -- is for HPV-6bE1; -- is for HPV-11E1. Rz1198 concentration is 0.7 nmol/L; two-substrate concentrations are 22.4 nmol/L, 11.2 nmol/L, 5.6 nmol/L, 2.8 nmol/L, 1.4 nmol/L for every dot from left to right; reaction volume: 5 L, incubated at 50 for 10 min.

4 Discussion

In 1981, Cech et al^[9] reported the discovery of the catalytic RNA (named by the authors as ribozyme), that possessed a sequence-specific catalytic activity on RNA. In 1986, Symons^[10] put forward the hypothesis of hammerhead type ribozyme, that was a hammerhead structure consisted of three helixes and a single-strand catalytic center. Since this kind of ribozyme was relatively simple in primary and secondary structures, and could be designed and synthesized in a laboratory, it aroused the interests of scientists and was widely employed^[11]. Our experiment was the study of preparation and cleavage activity of hammerhead ribozymeRz1198 by means of the computer design, cloning the ribozyme gene into the vector that possessed cis-cleavage ribozymes and labeled it with isotope. The in vitro transcription effect of Rz1198 was satisfactory, 5-cis-Rz and 3-cis-Rz cut themselves nine tenths already and released the purpose ribozyme. The ribozyme flanking sequences could be shortened, and the affection on ribozyme structure induced by the secondary structure of long flanking sequences would be eliminated. That would affect ribozyme turnover ratio and/or binding activity in the result of an accurate hybridization and better cleavage for ribozyme. The isolation of ribozyme was very convenient too. So, the vector possesses applicability in the study and in vitro preparation of ribozyme. Based on initial experiment (II), we affirmed that 5-cis-Rz and 3-cis-Rz did not disturb the cleavage of Rz1198; the in vitro study was carried out with labeled Rz1198. The authors believe that the method could easily obtain purified ribozyme, as well as make the Rz:S calculation and the kinetic constant estimation more accurate.

The kinetic results showed that Rz1198 from in vitro transcription possessed higher activity of in vitro cleavage, the optimal temperature was 50, the cleavage efficiency increased with the temperature heightened within the allowing temperature range, and Rz1198 possessed the property of a common enzyme. The values of k_m and k_cat of HPV-6bE1 and HPV-11E1 were similar, that could meet the need of the universality of Rz1198 for two substrates. Rz1198 could express good cleavage activity under physiological temperature at higher concentration. These results made Rz1198 to be worthy of being studied and developed as a nucleic acid drug.

However, the in vitro results cannot completely reflect its in vivo performance. The substrates we used was a part of HPV-6b/11E1 mRNA. The secondary and tertiary structures of the mRNAs transcribed from 2 kb HPV-6b/11E1 ORF would probably affect Rz1198 combination with the substrate and the cleavage activity. The expression and stability of Rz1198 in vivo needs to be searched too. In order to study Rz1198 expression and cleavage efficiency in vivo, the E1 gene with cis-acting ribozyme genes would be cloned into eucaryotic vectors and transfected into C127 cells. The investigation of Rz1198 function in vivo is under way.

5 Acknowledgments

The authors are grateful to Miss Chen L, Miss Liu J, Mr Xu F and Mr Zhou WX for their kind assistance.

References

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[7] Hughs F, Romanos M. E1 protein of human papillomavirus is a DNA helicase/ATPase. Nucleic Acids Res 1993; 21: 5817-23.
[8] Uhlenbeck OC. Small catalytic oligoribonucleotide. Nature 1987; 328: 596-600.
[9] Cech TR, Zang AJ, Grabowski PT. In vitro splicing of the ribosomal RNA precursor of tetrahymena: involvement of a guanine nucleotide in the excision of the intervening sequence. Cell 1981; 27: 487-96.
[10] Symons R. Self-cleavage of RNA in the replication of small pathogens of plants and animals. TIBS 1989; 14: 445-50.
[11] Bratty J, Chartrand P, Ferbeyre G. The hammerhead RNA domain, a model ribozyme. Biochem Biophys Acta 1993; 1216: 345-59.

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Project supported by the National Natural Science Foundation of China (39700129), Chinese Academy of Sciences (KJ951-B1-610) and Natural Science Foundation of Guangdong Province. Drs D.Z. Liu and Y.X. Jin are all first authors.
Correspondence to Dr. Y.Z. Huang. Dept. of Biochemistry, First Military Medical University, Guangzhou 510515, China.
e-mail: huangyangzhong@yahoo.com
Received 1999-10-06 Accepted 1999-11-10