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Ju-Sheng
Lin, Nan-Zhi Liu, Xin-Juan Kong, Na Xie, Nan-Xia Wang, Kuo-Huan
Liang, Institute of Liver Diseases, Tongji Hospital, Tongji
Medical College, Huazhong University of Science and Techology, Wuhan
430030,Hubei Province,China
Yu-Hu Song, You-Xin Jin, State Key Laboratory of Molecular
Biology, Shanghai Institute of Biochemistry, Chinese Academy of
Science, Shanghai 200031,China
Supported by Ministry of Health (No 98-1-140) and Chinese
Academy of Sciences (No KJ951-B1-610)
Correspondence to: Dr. JIN You-Xin, State Key Laboratory of
Molecular Biology, Shanghai Institute of Biochemistry, Chinese
Academy of Science, Shanghai 200031,China. yxjin@sunm.shcnc.ac.cn
Telephone: +86-21-64374430-221
Received 2001-07-19 Accepted 2001-10-28
Abstract
AIM:
To
study the preparation and cleavage activity of HpRz directed against
the transcript of HBV core gene in vitro.
METHODS:
HpRz gene designed by computer targeting the transcript of HBV core
gene was cloned into the vector p1.5 between 5’-cis-Rz and
3’-cis-Rz. 32p-labeled HpRz transcript proved whether
the vector fit for the preparation of hairpin ribozyme in vitro.
32p-labeled pKC transcript containing HBV core region as
target-RNA was transcribed using T7 RNA polymerase and
purified by denaturing PAGE. Cold HpRz transcript was incubated with
32p-labeled target-RNAs under different conditions and
radio autographed after denaturing polyacrylamide gel
electrophoresis.
RESULTS:
HpRz
has the specific ability of cleavage of target RNA at 37℃
and 12 mM MgCL2. Km=26.31nmol/L, Kcat=0.18/min.
These results revealed that the design of HpRz was correct.
CONCLUSION:
HpRz prepared in this study possesses specific catalytic activity
from the identification of cleavage activity. These results indicate
that hairpin ribozyme may intracellularly inhibit the replication of
HBV, therefore it may become a novel potent weapon for the treatment
of hepatitis B.
Song
YH, Lin JS, Liu NZ, Kong XJ, Xie N, Wang NX, Jin YX, Liang KH. Anti-HBV
hairpin ribozyme-mediated cleavage of target RNA in vitro .
World J Gastroenterol 2002;8(1):91-94
INTRODUCTION
Hepatitis
B is a major worldwide health problem[1-5]. Hepatitis B
virus is a small hepatotropic DNA virus, causing acute and chronic
B-type hepatitis in man. Chronic infection is associated with a high
risk of liver cirrhosis and primary liver carcinoma[6-14].
Currently available therapies are of limited efficacy[15-33].
Ribozyme is a kind of catalytic RNA which can catalyze the cleavage
of sequence-specific RNA. Compared with antisense RNA, ribozyme may
be a more effective experimental tool to suppress the gene
expression. Possessing both antisense and RNA cleavage activity, the
enzymatic nature of ribozyme may facilitate effectiveness even at
low level of expression[34]. Hepatitis B virus is a DNA
virus that replicates through reverse transcription of a RNA
intermediate (pregomic RNA). So engineered anti-HBV ribozyme can
potentially be multifunctional, targeting pregenomic RNA and viral
mRNA to suppress the replication of hepatitis B virus. Since
previous attempt to use hammerhead ribozyme for the intracellular
inhibition of HBV replication have been largely unsuccessful in
intact cell[35,36], however engineered hairpin ribozymes
have been shown to inhibit replication of human immunodeficiency
virus, hepatitis C virus and human papilloma virus in vivo [37-44].
So we designed hairpin ribozyme to inhibit HBV replication. It is
well known that HBV C region is associated with viral replication.
In this report we have designed hairpin ribozyme targeting core gene
of HBV by computer, prepared hairpin ribozyme by transcription of
recombinant plasmid in vitro , and proved its ability to
cleave HBV in vitro from identification of activity of anti-HBV
hairpin ribozyme.
MATERIALS
AND METHODS
Materials
E. coli DH5α has been maintained in our laboratory. The
ribozyme vector p1.5 (kind gift of Qi GR, Shanghai Institute of
Biochemistry), Plasmid pKC[45] for target-RNAs
(constructed by Dr Lian JQ, the Forth Military Medical University)
has been maintained in our laboratory. DNA sequence Kit, in vitro
transcription kit, restriction endonucleases, T4 DNA ligase,
RNase A free DNase I were purchased from Promega Company; and RNase
inhibitor, T4 DNA polymerase from Takara Company, α 32p
dATP and α 32 UTP from Beijing Yahui Company,
Materials were used all of analytical purification.
Oligonucletides of HpRz: R1 5’-CTA GAT CCG GAA GAA CTA AAC
CAG AGA AAC AGA TCT CTT CGG AGA TCG TAC ATT ACC TGG TAG GTA C-3’;
R2 5’-CTA CCA GGT AAT GTA CGA TCT CCG AAG AGA TCT GTT TCT CTG GTT
TAG TTC TTC CGG AT-3’. They were chemically synthesized in Beckman
Oligo-1000 DNA synthesizer.
Methods
In vitro transcription and purification of target RNA The
template PKC was linearized with BstXI, the 3’-overhangs of
linearized pKC was blunted with T4 DNA polymerase. in vitro transcription
was carried out at 37℃
for 90 min in a 20 μL final volume containing 40 mmol·L-1
Tris·Hcl (pH 7.5),50 mmol·L-1 DTT, 2 mmol·L-1
spermidine,8 mmol·L-1 Mgcl2, 0.25 mmol·L-1
ATP, GTP,CTP, 0.05 mmol·L-1UTP,370 KBq alpha 32p-UTP,80
U T7 RNA polymerase and 4 μg linearlized template.
Target RNA was purified by 60g·L-1 denaturing gel
electrophoresis by cutting off the autoradiograph bands. Labeled
target RNA was dissolved in DEPC H2O and reserved under
-20℃.
Construction of recombinant plasmid for ribozyme and preparation
of ribozyme in vitro The hairpin ribozyme HpRz for HBV
was designed according to the computer software pcFOLD combiled by
Professor Zuker (Canadian Academy of Science). The homologous
possibility with the gene of human being was excluded by consulting
with RNA sequence of human cell from NCBI Genbank. Synthesized
ribozyme fragments were cloned into XbaI/KpnI sites of p1.5 as pHpRz.
Recombinant plasmid was identified by being digested with EcoRI and
HindIII, then the DNA sequence of recombinant plasmid was analyzed
through the dideoxy method developed by Sanger(Figure 1). The
template pHpRz was linearized with EcoRI and in vitro transcription
and purification of ribozyme were the same as those of target RNA.
To get a large amount of transcribed HpRz without isotope, the
transcription of HpRz was set up according to manufacture’s
instruction, then template was digested with RNAse A free DNase I,
the RNA was dissolved in DEPC H2O and measured by
spectrophotomer containing 3’-cis-Rz and 5’-cis-Rz.
Figure
1(PDF) Structure
of HpRz plus 5’-cis-Rz and 3’-cis-Rz.
in vitro cleavage reaction of HpRz The cold
hairpin ribozyme and target RNA labeled by alpha- 32p UTP
were quantified. The cleavage reaction of ribozyme was performed in
ribozyme buffer (40 mmol·L-1 Tris·Hcl pH7.5/ 12 mmol·L-1
Mgcl2 ) at 37℃
with 10 nmol·L-1 32p-labeled
substrate and 10 n mol·L-1 cold ribozyme. In this
condition the cleavage mixture was incubated at different time
points.The volume of the reaction is 5μL.One μL loading
buffer (2.5g·L-1 Bromophenol Blue, 2.5g·L-1
Xylene cyanol FF, 20 mmol·L-1 EDTA and saturated Urea)
was added to stop the reaction. The result could be analyzed after
running a 60g·L-1 denaturing polyacylamide gel
electrophoresis. The cleavage efficiency was calculated from Bq
values of the bands of substrate (S) and products (P) CE=[P/(P+S)]·100%.
Kinetics of the cleavage reaction The procedure was described
by Uhlenbeck[46].The cleavage reactions were done and the
results were analyzed as above. Km and Kcat
were calculated by Lineweaver-Burk method (double- riciprocal plot
).
RESULTS
Identification
of recombinant plasmid pHpRz
The result of digested recombinant plasmid by EcoRI and HindIII was
analyzed by running 20g·L-1 agrose gel elrctropheries
(Figure 2).This result and the DNA sequence analysis showed that the
clone was correct.
Identification
of transcription of ribozyme
The transcription of ribozyme from EcoRI-linearized template should
include three bands: 50nt, 63nt and 83nt. The resultshowed our
design was correct (Figure 3).
Figure
2
The analysis of HpRz digested by EcoRI and HindIII(20g·L-1
agrose gel). lane 1:λ DNA/HindIII marker; lane 2: DL2000 DNA
marker; lane 3-7: HpRz digested by EcoRI and HindIII.
Figure
3 in
vitro transcript
of pHpRZ. HpRz is 83nt. 5’-cis-Rz is 63nt, 3’-cis-Rz is 50nt.
In
vitro cleavage reaction of ribozyme and kinetics of the cleavage
reaction
Time course The cleavage mixture ( Rz :substrate=1:1 mol·L-1)were
incubated at 37℃
for different time points,it was shown that the reaction product
increased with the increase in incubation time and within 60 min it
was linear, CE=32.67%(Figures 4 and 5 ).
Figure
4 Time
course. Specific cleavage of HBV RNA target molecules by HpRz in
vitro . Lane 1:substrate control; lane 2:incubated for 6 min;
lane 3: 10 min; lane 4:20 min; lane 5:40 min; lane 6:60 min; lane
7:90min.
The
kinetics of cleavage reaction Under
the condition of 37℃
and 40-minute reaction time, the cleavage efficiency was calculated
at Rz:S=1:1,1:2,1:4,1:8 and 1:16(mol/L)ratio. Km and Kcat
were obtained by the Lineweaver-Burke method (Figure 6) Km
=26.31nmol·L-1 , Kcat =0.18·min-1.
Figure
5(PDF)
Time curves of cleavage reactions of HpRz prepared in vitro.
Figure 6(PDF)
Lineweaver-burk
kinetic plots of the specific cleavage of HBV RNA target molecules
by HpRz prepared in vitro. HpRz concentration is 5 nmol·-1,
substate concentration is 80,40,20,10 5nmol·L-1.
Reaction is at 37℃
for 40 min.
DISCUSSION
Ribozyme
is classified into six kinds. Hammerhead ribozyme and hairpin
ribozyme have been studied extensively as experimental tools for
trans suppression of gene expression and possible therapeutic
application. Hammerhead ribozyme is small and easy to design and
there is less restriction of selection of target site, so its
application is extensive. Although hammerhead ribozyme was used to
suppress HBV replication, and hammerhead ribozyme-mediated cleavage
of target RNA could be achieved in vitro and in cell extract,
they showed poor effect in intact cells[35,36]. Cellular
protein and low Mg2+ concentration limited intracellular
ribozyme activity. It will be difficult to raise the intracellular
Mg2+ level. Protein can influence the ribozyme
intracellular performance. However, cellular protein may also have
positive effect on ribozyme-mediated catalysis. Because of limited
knowledge on mechanisms of protein modulation of catalytic RNA
activity and the complex formed by ribozyme and ribonucleoprotein
are frequently large, poorly defined and difficult to study, it is
hard for us to use protein to enhance ribozyme activity. Compared
with hammerhead ribozyme, hairpin ribozyme has complex structure,
hard to design and there is too much restriction of target site and
its application is limited. It possesses the better cleavage
activity under physiological condition from in vitro assays
done under standard condition at 37℃
and low Mg2+ concentration. PJ Welch[47]
designed three hairpin ribozymes against the pgRNA (pregomic RNA)
encoding HBV surface antigen, the polymerase and the X protein and
demonstrated that two of them can cleave target RNA in vitro and
reduced the level of HBV expression and suppressed HBV replication in
vivo . Zu et al [48] used hairpin ribozyme
library to identify accessible target sites within HBV progenomic
RNA, four hairpin ribozyme targeting conserved region in subtypes of
HBV can inhibit HBV replication in intact cell. Until now no report
on hairpin ribozyme directed against core gene of HBV has available.
In this report we have designed and constructed hairpin ribozyme to
cleave transcripts of HBV core gene in vitro . Our experiment
was the study of preparation and cleavage activity of hairpin
ribozyme-HpRz 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
ribozyme was satisfactory, 5’-cis-Rz and 3’-cis-Rz cut
themselves and released the purposed ribozyme. The ribozyme flanking
sequences could be shortened and ribozyme structure induced by the
secondary structure of long flanking sequences would be eliminated.
That would affect ribozyme turnover ratio/binding activity in the
result of an accurate hybridization and better cleavage for ribozyme.
Moreover isolation of ribozyme is very convenient by cutting off
autoradiograph bands. Ribozyme is quantified more accurately.In this
paper the number of nucleotide of hairpin ribozyme was similar to
that of the cleavage product, so we can not use transcribed HpRz
labeled by isotope. The ribozyme vector was proved to fit for
preparation of hammerhead ribozyme[49-51], In our study
we also used the preparation of hairpin ribozyme and its efficacy is
similar to that of hammerhead ribozyme. So the vector possesses wide
applicability in the study of preparation of ribozyme in vitro ,not
only hammerhead ribozyme, but also hairpin ribozyme.
HBV is a double-stranded DNA virus replicates through
pregenomic RNA intermediate, which provides a therapeutic
opportunity for a novel antiviral gene therapy based on ribozyme RNA
cleavage. In our experiment, we have designed and constructed
hairpin ribozyme directed against HBV core region. The kinetics of
hairpin ribozyme showed that HpRz possessed specific ability of
cleaving the HBV transcripts in vitro . These results
indicated that HpRz is worthy of being further studied in intact
cells and developed as a nucleic acid drug in the future. However
the in vitro result can not completely reflect in vivo
performance. The total HBV mRNA transcript in cell forms the
secondary and tertiary structure which affect ribozyme combination
with the substrate and cleavage activity. The subcellular
compartment where the ribozyme is located, degradation of ribozyme,
the complexes which are formed by ribozyme and ribonucleoprotein
within cell and gene delivery system, affect the combination with
substrate and cleavage of ribozyme. So in vivo effect of the
ribozyme should be investigated as soon as possible.The ribozyme
fragments would be cloned into eucaryotic vectors and transfected
into HepG2 cell or Huh7 cell. Experimental analysis of the anti-HBV
ribozyme activity in vivo is in progress and should help
determine its potential use as antiviral agents against HBV.
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