[Cleavage of in vitro transcripts of hepatitis B virus C gene by 10-23 DNA enzyme].
To investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro.



10-23 DNA enzymes named DrzBC-7, DrzBC-8 and DrzBC-9 specific to HBV C gene ORF A1816UG were designed and synthesized. HBV C gene mRNA was obtained by in vitro transcription method. Cleavage activities were observed in vitro. The influence of MgCl2 concentration on RNA cleaving activity was examined with DrzBC-9. Values of kinetic parameters including Km, Kcat and Kcat/Km were calculated accordingly.



Targeted substrate mRNA with the size of 300 nt was obtained by transcription in vitro. Under the certain cleavage conditions, DrzBC-7, DrzBC-8 and DrzBC-9 all efficiently cleaved target mRNA at specific sites in vitro. Cleavage products of 109 nt and 191 nt were obtained. No cleavage occurred without MgCl2. The most efficient cleavage was obtained at 150 mmol x L(-1) MgCl2. The efficiency of cleavage did not increase when the MgCl2 concentration was more than 200 mmol x L(-1). The kinetic parameters, Km, Kcat and Kcat/Km for DrzBC-9 were 1.4x10(-9) mol x L(-1), 1.6 min(-1) and 1.1x10(9) mol x L(-1) x min(-1), respectively.



10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.
He
Wei
W
Institute of Infectious Diseases, The First Affiliated Hospital of Medical College, Zhejiang University, Key Laboratory of Infections Diseases, Ministry of Public Health, Hangzhou 310003, China.
Wo
Jian-er
JE
Liu
Ke-zhou
KZ
D021881:DNA, Catalytic / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D006515:Hepatitis B virus / Q000201:enzymology / Q000235:genetics*; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D012367:RNA, Viral; C402582:RNA-cleaving DNA 10-23