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. 2002 Feb;8(1):91-4.
doi: 10.3748/wjg.v8.i1.91.

Anti-HBV hairpin ribozyme-mediated cleavage of target RNA in vitro

Yu-Hu Song  1 , Ju-Sheng Lin, Nan-Zhi Liu, Xin-Juan Kong, Na Xie, Nan-Xia Wang, You-Xin Jin, Kuo-Huan Liang
Affiliations

Anti-HBV hairpin ribozyme-mediated cleavage of target RNA in vitro

Yu-Hu Song et al. World J Gastroenterol. 2002 Feb.

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 degrees and 12 mM MgCl2. Km=26.31 nmol/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.

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Figures

Figure 1
Figure 1
Structure of HpRz plus 5’-cis-Rz and 3’-cis-Rz.
Figure 2
Figure 2
The analysis of HpRz digested by EcoRI and HindIII(20 g·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
Figure 3
in vitro transcript of pHpRZ. HpRz is 83nt. 5’-cis-Rz is 63nt, 3’-cis-Rz is 50nt.
Figure 4
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 7:90 min.
Figure 5
Figure 5
Time curves of cleavage reactions of HpRz prepared in vitro.
Figure 6
Figure 6
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 5 nmol·L-1. Reaction is at 37 °C for 40 min.
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