Selection of DNA aptamers that bind the RNA-dependent RNA polymerase of hepatitis C virus and inhibit viral RNA synthesis in vitro.
The RNA-dependent RNA polymerase (NS5B) of the hepatitis C virus (HCV) plays a key role in the life cycle of the virus. In order to find inhibitors of the HCV polymerase, we screened a library of 81 nucleotide (nt)-long synthetic DNA containing 35 random nucleotides by the Systematic Evolution of Ligands by Exponential enrichment (SELEX) approach. Thirty ligands selected for their binding affinity to the NS5B were classified into four groups on the basis of their sequence homologies. Among the selected molecules, two were able to inhibit in vitro the polymerase activity of the HCV NS5B. These aptamers appeared to be specific for HCV polymerase, as no inhibition of poliovirus 3D polymerase activity was observed. The binding and inhibitory potential of one aptamer (27v) was associated with the 35 nt-long variable region. This oligonucleotide displayed an apparent dissociation constant (K(d)) in the nanomolar range. Our results showed that it was able to compete with RNA templates corresponding to the 3'-ends of the (+) and the (-) HCV RNA for binding to the polymerase. The fact that a DNA aptamer could interfere with the binding of natural templates of the enzyme could help in performing structure-function analysis of the NS5B and might constitute a basis for further structure-based drug design of this crucial enzyme of HCV replication.
Bellecave
Pantxika
P
UMR 5097 CNRS, Institut Fédératif de Recherches Pathologies Infectieuses et Cancer (IFR 66), Université Victor Segalen Bordeaux 2, 33076 Bordeaux cedex, France.
Andreola
Marie-Line
ML
Ventura
Michel
M
Tarrago-Litvak
Laura
L
Litvak
Simon
S
Astier-Gin
Thérèse
T
D001483:Base Sequence; D024202:Electrophoretic Mobility Shift Assay; D015723:Gene Library; D016174:Hepacivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase