[Study on the cleavage activity of U1 small nuclear RNA chimeric ribozyme against HCV RNA in vitro].
OBJECTIVE
To study the cleavage activity on the HCV RNA of a chimeric recombinant of HCV specific ribozyme and U1 small nuclear RNA, which compartmentalizes within the nucleolus.


METHODS
The third stem-loop sequence of human U1 snRNA (position 95-116) within pBSIISK+ U1 was substituted by hammerhead ribozyme against HCV RNA by PCR and cloning methods, and the constructed plasmid was named pBSIISK+ (U1-Rz). Then the whole gene fragment of the chimeric ribozyme was cloned into a pGEM-T vector under the control of T7 promoter, and the constructed plasmid was named pGEM- (U1-Rz). The pGEM- (U1-Rz) and pGEM-Rz (containing the same ribozyme sequence as that in U1-Rz) transcripts as enzyme were transcribed in vitro. Also the (32)P-labeled pCMV/T7-NCRC luc (containing the gene sequence of the whole 5'-NCR and part core of HCV RNA) transcripts as target-RNAs were transcribed in vitro. The enzymes were incubated with the target RNAs under different conditions and autoradiographed after denaturing gel-electrophoresis.


RESULTS
The sequencing result showed that the construction of U1 snRNA chimeric ribozyme was correct. Compared with the ribozyme alone, both of them were active at 37 degree C and with Mg2+ (10 mmol/L) and TrisCl (10 mmol/L, pH7.9), and there was no remarkable difference between them. The cleavage activity of the chimeric ribozyme increased with the prolongation of reaction time and increment of enzyme concentration.


CONCLUSIONS
Both ribozyme and U1 snRNA chimeric ribozyme exhibited specifically catalytic activity against HCV RNA in vitro. There was no remarkable difference between their cleavage efficiencies.
Tian
Mei-Mei
MM
Beijing Ditan Hospital, Beijing 100011, China.
Wang
Feng
F
Niu
Jun-Qi
JQ
Wang
Mei-Xia
MX
D002678:Chimera / Q000235:genetics; D015316:Genetic Therapy; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000628:therapy; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*
D016337:RNA, Catalytic; D012342:RNA, Small Nuclear; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; C405483:U1 small nuclear RNA; C427204:hammerhead ribozyme