A nonisotopic assay method for hepatitis C virus NS5B polymerase.
The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is responsible for replication of genomic RNA. A novel nonisotopic assay method is described for detecting its enzymatic activity. The 5' end of the in vitro-transcribed template RNA was attached covalently to the surface of a Covalink module using carbodiimide condensation. The RNA strand containing the 3' untranslated region (3' UTR) of HCV at its 3' end was free in the solution. A purified NS5B polymerase and NTPs along with biotin-labeled UTP were added to this module and the polymerization activity could be detected colorimetrically with streptavidin-conjugated alkaline phosphatase.
Park
Chanhee
C
Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Kyung-Gi Do, South Korea.
Kee
Younghoon
Y
Park
Jungchan
J
Myung
Heejoon
H
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000469:Alkaline Phosphatase / Q000737:chemistry; D001665:Binding Sites; D019921:Biotinylation; D002234:Carbodiimides / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D005912:Gliotoxin / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007700:Kinetics; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000032:analysis*; D011868:Radioisotopes; D019809:Streptavidin / Q000737:chemistry; D013698:Templates, Genetic; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000032:analysis*
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D002234:Carbodiimides; D007166:Immunosuppressive Agents; D012367:RNA, Viral; D011868:Radioisotopes; D017361:Viral Nonstructural Proteins; D005912:Gliotoxin; D019809:Streptavidin; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; D000469:Alkaline Phosphatase