Aim: To study the preparation and cleavage activity of Rz1196 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 ...Aim: To study the preparation and cleavage activity of Rz1196 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 ofT7 promoter, ～(32)P-labeted 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 between5'-cis-Rz and 3'-cis-Rz. a2P-labeled Rzl198 transcript was gel-purified, incubated with target-RNAs at different condi-tions and autoradiographed after denaturing gel-electrophoresis. Results: Rz1198 was active at 37℃. The optimaltemperature 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 thetwo substrates--HPV-6bE1 and HPV-11E1 transcripts. Conclusion: Rz1198 prepared in vitro possesses the perfectspecific catalytic cleavage activity. It leads to the expectation that, in the future, it will be possible to develop a newnucleic acid drug from Rz1198 which can efficiently inhibit the replication of HPV-6b/11 DNA in vivo. (Asian J An-drol 1999 Dec; 1: 195-201)展开更多
文摘Aim: To study the preparation and cleavage activity of Rz1196 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 ofT7 promoter, ～(32)P-labeted 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 between5'-cis-Rz and 3'-cis-Rz. a2P-labeled Rzl198 transcript was gel-purified, incubated with target-RNAs at different condi-tions and autoradiographed after denaturing gel-electrophoresis. Results: Rz1198 was active at 37℃. The optimaltemperature 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 thetwo substrates--HPV-6bE1 and HPV-11E1 transcripts. Conclusion: Rz1198 prepared in vitro possesses the perfectspecific catalytic cleavage activity. It leads to the expectation that, in the future, it will be possible to develop a newnucleic acid drug from Rz1198 which can efficiently inhibit the replication of HPV-6b/11 DNA in vivo. (Asian J An-drol 1999 Dec; 1: 195-201)
