Design and activity assessment of HDV ribozymes to trans-cleave HCV RNA
10.3321/j.issn:1000-5404.2001.03.019
- VernacularTitle:剪切HCV RNA的HDV核酶的设计及活性测定
- Author:
Yue-Cheng YU
1
;
Chang-Hai GU
;
Qing MAO
;
Qi-Fen LI
;
Yu-Ming WANG
;
Huan-Zhen GUO
Author Information
1. 第三军医大学附属西南医院
- From:Journal of Third Military Medical University
2001;23(3):312-314
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the probability of using hepatitis D virus (HDV) ribozyme as a kind of anti-hepatitis-C-virus (HCV) gene thera-py drugs. Methods The natural HDV genomic ribozyme′s stem Ⅳ was optimized and its substrate-binding region reconstructed, thus three recombinant HCV-specific HDV genomic ribozymes RzC1, RzC2 and RzC3 were obtained. HCV RNA 5'-noncoding region and 5'-fragment of C region (HCV RNA5'-NCR-C) were transcribed from plasmid pHCV-neo by T7 phage RNA polymerase in vitro, and radiolabelled at its 5'-end. The trans-cleaving reaction was performed by mixing the ribozymes and substrate at mol ratio 100∶1 under conditions as follows: 37℃, pH7.5, Mg2+ 20 mmol/L and deionized formamide 2.5 mol/L. Percentage of trans-cleaved products were calculated at different time points and used as the activity indicator of the three ribozymes. Results RzC1, RzC2 trans-cleaved more substrate when the time extended, and got to 24.9%,20.3% after reac-ting for 90 minutes respectively; RzC3 was not able to trans-cleave its substrate. Conclusion Recombinant HDV genomic ribozymes have the ability to trans-cleave HCV RNA, but the appropriate target sequence should be selected.
