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Volume 1, Issue 4 (December 1999) 1, 195–201;
Preparation and identification of activity of anti-HPV-6b/11E1 universal ribozyme--Rz1198 in vitro
D.Z. Liu, Y.X. Jin, H. Hou, Y.Z. Huang, G.C. Yang, Q. Xu
1.State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai 200031,China
2.Department of Biochemistry- First Military Medical University, Guangzhou 510515, China
Advance online publication 1 December 1999
| Abstract |
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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, km=12.2 nmol/L, kcat=0.18 min-1; For HPV-11E1, km=14.7 nmol/L, kcat=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
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