[Inhibition of hepatitis B virus S gene and C gene expression by different 10-23 DNAzymes substrate-recognition domains].
OBJECTIVE
To explore the inhibition effects of 10-23 DNAzymes with different substrate-recognition domains targeting hepatitis B virus (HBV) S gene and C gene expression in 2.2.15 cells.


METHODS
10-23 DNAzymes with different substrate-recognition domains specific to HBV S gene open reading frame (ORF) A(157)UG and HBV C gene ORF A(1816)UG were designed and synthesized, respectively. Different 10-23 DNAzymes were transfected into 2.2.15 cells which is a stable HBV producing cell line. HBsAg and HBeAg secreted into culture media were detected by radioimmunoassay (RIA) and HBV DNA levels were measured by real-time PCR. 3-(4, 5-dimethylthiagol-2-yl)-2, 5-drphnyl tetrazolium bromide (MTT) assays were performed to evaluate cytotoxicity.


RESULTS
HBsAg and HBeAg expressions were reduced by various DNAzymes (0.1 - 2.5 micromol/L) with different substrate-recognition domains after transfection. The antiviral effects of DNAzymes were apparent until 72 h post-transfection. The inhibition rates of the DNAzymes at the same dose on HBsAg and HBeAg in the same period of post-transfection were as the following: DrzBS-9 > DrzBS-8 > DrzBS-7; DrzBC-9 > DrzBC-8 > DrzBC-7. Among all the DNAzymes used, DrzBS-9 targeting S gene and DrzBC-9 targeting C gene were most potent, with HBsAg and HBeAg reduced 95% and 92% 48 h post-transfection at the dose of 2.5 micromol/L, respectively. The inhibition effects on HBV DNA by various DNAzymes with different substrate-recognition domains were of no significance. There were no evident cytotoxic effects of these DNAzymes in the range from 0.1 to 2.5 micromol/L.


CONCLUSIONS
10-23 DNAzymes with different substrate-recognition domains targeting HBV S gene and C gene mRNA possessed specific inhibition effects in 2.2.15 cells, and DrzBS-9 targeting S gene and DrzBC-9 targeting C gene were most potent.
Hou
Wei
W
Institute of Infectious Diseases, First Affiliated Hospital, College of Medical Sciences, Zhejiang University, Hangzhou 310003, China.
Wo
Jian-er
JE
Liu
Ke-zhou
KZ
Li
Min-wei
MW
Chen
Li-wei
LW
Hu
Zhong-rong
ZR
Liu
Rong-hua
RH
Hu
Min-jun
MJ
D002460:Cell Line; D021881:DNA, Catalytic / Q000494:pharmacology*; D004279:DNA, Viral / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D005814:Genes, Viral; D006514:Hepatitis B Surface Antigens / Q000032:analysis; D006513:Hepatitis B e Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D014162:Transfection
D021881:DNA, Catalytic; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens