In vitro cleavage of hepatitis B virus C mRNA by 10-23 DNA enzyme.
BACKGROUND
10-23 DNA enzyme is one kind of deoxyribozymes for RNA cleavage. The inhibition effects of 10-23 DNA enzyme on the expression of the HBV C gene in HepG2.2.15 cells were demonstrated previously. The aim of this study was to further explore the cleavage activities of 10-23 DNA enzyme targeting at HBV C gene mRNA in vitro.


METHODS
10-23 DNA enzyme named Drz-HBV-C-9 specific to HBV C gene ORF A(1816)UG was designed and synthesized. HBV C gene mRNA was obtained by the in vitro transcription method. Cleavage activities of Drz-HBV-C-9 were observed in vitro. Values of kinetic parameters including Km,Kcat and Kcat/Km were calculated accordingly.


RESULTS
Under the certain cleavage conditions, Drz-HBV-C-9 could efficiently cleave target mRNA at specific sites in vitro. Cleavage products of 109nt plus 191nt were obtained. The kinetic parameters, Km, Kcat and Kcat/Km for Drz-HBV-C-9, were 1.4 X 10(-9) mol, 1.6 min-1 and 1.1 X 10(9) mol(-1) x min(-1), respectively.


CONCLUSIONS
10-23 DNA enzyme targeting at HBV C gene mRNA possesses specific cleavage activities in vitro. This would be a potent antiviral strategy with respect to HBV gene therapy.
Hou
Wei
W
Institute of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
Wo
Jian-Er
JE
Li
Min-Wei
MW
Liu
Ke-Zhou
KZ
D000998:Antiviral Agents; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D017931:DNA Primers; D021881:DNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D006515:Hepatitis B virus / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D000998:Antiviral Agents; D003062:Codon; D017931:DNA Primers; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D012367:RNA, Viral; C402582:RNA-cleaving DNA 10-23