Inhibition of hepatitis C virus serine protease in living cells by RNA aptamers detected using fluorescent protein substrates.
Hepatitis C virus is one of the causative agents of non-A non-B hepatitis. Since one of viral proteins, NS3, has serine protease activity indispensable for virus maturation. NS3 serine protease is considered to be a suitable target for anti-HCV reagents. We report an assay of HCV NS3 protease in living cells. We designed peptide substrates bearing one of the sequences of HCV NS3 protease cleavage sites sandwiched with fluorescent proteins CFP and YFP. Substrates were expressed and cleaved efficiently in HeLa cells by cotransfection with HCV NS3 protease. The relationship between the progress of cleavage reaction and the change in fluorescence of the substrate emitted from living cells was confirmed. As a group of candidates for inhibitor of HCV NS3 protease, we chose RNA aptamers, nucleic acid ligands selected from a completely random RNA pool by in vitro selection. We found that 3 classes of aptamers, G9-I, II and III, bound NS3 protease specifically and inhibited cleavage in vitro. We studied the effect of RNA aptamers introduced into HeLa cells. The addition of G9-II RNA in the medium at a concentration of 2.5 micro g/ml reduced cleavage by one-third that of control.
Kakiuchi
N
N
Institute of Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan. kakiuchi@p.kanazawa-u.ac.jp
Fukuda
K
K
Nishikawa
F
F
Nishikawa
S
S
Shimotohno
K
K
D015153:Blotting, Western; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006526:Hepatitis C / Q000201:enzymology*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*
D005456:Fluorescent Dyes; D015842:Serine Proteinase Inhibitors; D012313:RNA