[Activity of specific deoxyribozymes to cleave hepatitis C virus RNA in vitro].
OBJECTIVE
To analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy.


METHODS
Two specific sequences containing 5' ...A / U... 3' in HCV 5'-noncoding region and 5'-fragment of C region (5'-NCR-C) were selected as the target sites, and with the active region of 5'GGCTAGCTACAACGA3', two phosphorothioate deoxyribozymes (TDRz) named as TDRz-127 and TDRz1 were synthesized. HCV RNA 5'-NCR-C was transcribed in vitro from plasmid pHCV-neo which was completely linearized with restriction endonuclease Nar I, and its 5'-end phosphoric acid was deleted by calf intestinal alkaline phosphatase (CIP), then radiolabelled with T4 polynucleotide kinase and gamma-32P-ATP. Under the conditions such as pH 7.5 and a 10 mmol/L Mg2+ concentration, TDRz-127 and TDRz1 were separately (a 5 micromol/L final concentration) or combinedly (each 2.5 micromol/L) mixed with the substrate RNA (200 nmol/L). After denaturation and then renaturation, the reaction systems were incubated in 37 degrees C, and aliquots were removed to terminate the reaction at intended time points. The cleavage products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. Finally, the optical density of each product band was measured with Gel Documentation-Analyzing Systems for calculating the percentages of cleaved HCV 5'-NCR-C.


RESULTS
After reaction for 15, 30, 45, 60, 75 and 90 min under the adopted conditions, about 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1% of HCV 5'-NCR-C was cleaved by TDRz-127 respectively; 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3% by TDRz1; and 15.1%, 29.6%, 37.8%, 39.1%, 41.5%, 42.6% by combining the two TDRzs.


CONCLUSIONS
Cleavage percentage of both TDRz-127 and TDRz1 increases with the time, and the effect of combining the two TDRzs is better than that of anyone.
Yu
Yue-cheng
YC
Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Gu
Chang-hai
CH
Wang
Sheng-qi
SQ
Mao
Qing
Q
Chen
Zhong-bin
ZB
Liu
Hong-ling
HL
D020121:5' Untranslated Regions / Q000378:metabolism; D001483:Base Sequence; D021881:DNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics / Q000378:metabolism
D020121:5' Untranslated Regions; D021881:DNA, Catalytic; D016337:RNA, Catalytic; D012367:RNA, Viral; D039221:mRNA Cleavage and Polyadenylation Factors