Preparation and identification of activity of anti-HPV-6b/11E1 universal ribozyme--Rz1198 in vitro.
OBJECTIVE
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 degrees C. The optimal temperature was 50 degrees C. 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 substrates--HPV-6bE1 and HPV-11E1 transcripts.


CONCLUSIONS
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.
Liu
D Z
DZ
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences.
Jin
Y X
YX
Hou
H
H
Huang
Y Z
YZ
Yang
G C
GC
Xu
Q
Q
D027383:Papillomaviridae / Q000235:genetics*; D016337:RNA, Catalytic / Q000302:isolation & purification*; D014158:Transcription, Genetic
D016337:RNA, Catalytic