[Experimental study on HDV ribozyme in vitro cleaving the HBV derived RNA fragment].
OBJECTIVE
To explore the possibility of transacting hepatitis D virus (HDV) ribozyme cleaving in vitro the hepatitis B virus (HBV) mRNA fragments.


METHODS
According to the established pseudoknot-like structure, its' H1 domain was changed to design the transacting HDV ribozyme Rc1 and Rc2, which targeted the 701-713 site and 776-788 site of HBV C domain. After the chemically synthesised cDNA of the ribozyme was cloned into the vector PGEM-4Z, the transacting HDV ribozyme was transcriped using in vitro transcription technology. The in vitro cleavage characteristics of the ribozyme were studied and the kinetic parameters (Kcat and Km) were determined by Eadie Hofstee plotting.


RESULTS
Both the two ribozymes had the ability to cleave the substrate, the cleavage percentage at 37 degrees for 90 minutes were 50% and 51%. According to the Eadie Hofstee plot, the Km of the Rc1 and Rc2 were 0.61 micromol and 0.58 micromol, the Kcat were 0.64 x min(-1) and 0.60 x min(-1),respectively.


CONCLUSIONS
The cleaving ability of trans-acting HDV ribozyme on non-HDV RNA fragment was tested. The results showed a new potential of the antisense antisense regent for HBV gene therapy.
Pan
Guang-jin
GJ
Shandong Academy of Medical Sciences, Jinan 250062, China.
Han
Jin-xiang
JX
D016373:DNA, Antisense / Q000235:genetics; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics*; D003698:Hepatitis Delta Virus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D014158:Transcription, Genetic
D016373:DNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral