Activity of HDV ribozymes to trans-cleave HCV RNA.
OBJECTIVE
To explore whether HDV ribozymes have the ability to trans-cleave HCV RNA.


METHODS
Three HDV genomic ribozymes were designed and named RzC1, RzC2 and RzC3. The substrate RNA contained HCV RNA 5'-noncoding region and 5'-fragment of C region (5'-NCR-C). All the ribozymes and HCV RNA 5'-NCR-C were obtained by transcription in vitro from their DNA templates, and HCV RNA 5'-NCR-C was radiolabelled at its 5'-end. Under certain pH, temperature, appropriate concentration of Mg(2+) and deionized formamide, these ribozymes were respectively or simultaneously mixed with HCV RNA 5'-NCR-C and reacted for a certain time. The trans-cleavage reaction was stopped at different time points, and the products were separated with polyacrylamide gel electrophoresis (PAGE), displayed by autoradiography. Percentage of trans-cleaved products was measured to indicate the activity of HDV ribozymes.


RESULTS
RzC1 and RzC2 could trans-cleave 26 % and 21.8 % of HCV RNA 5'-NCR-C under our reaction conditions with 2.5 mol.L(-1) deionized formamide respectively. The percentage of HCV RNA 5'-NCR-C trans-cleaved by RzC1, RzC2 or combined usage of the three ribozymes increased with time, up to 24.9 %, 20.3 % and 37.3 % respectively at 90 min point. Almost no product from RzC3 was observed.


CONCLUSIONS
HDV ribozymes are able to trans-cleave specifically HCV RNA at certain sites under appropriate conditions, and combination of several ribozymes aiming at different target sites can trans-cleave the substrate more efficiently than using only one of them.
Yu
Yue-Cheng
YC
Institute of Infectious Diseases of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing 400038,China. yuechengyu212001@163.net
Mao
Qing
Q
Gu
Chang-Hai
CH
Li
Qi-Fen
QF
Wang
Yu-Ming
YM
D001483:Base Sequence; D016174:Hepacivirus / Q000235:genetics*; D003698:Hepatitis Delta Virus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D016337:RNA, Catalytic; D012367:RNA, Viral