title: Functional binding of hexanucleotides to 3C protease of hepatitis A virus.
abstract: Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C(pro)). Inhibition assays in vitro identified the hexanucleotide 5'-GGGGGT-3' (G(5)T) as a 3C(pro) protease inhibitor. Using (1)H NMR spectroscopy, G(5)T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of (1)H, (15)N-HSQC experiments the binding site for G(5)T was located to the C-terminal β-barrel of HAV 3C(pro). Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G(5)T-binding site, nor does G(5)T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid-protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.
authors.affiliation: Institute of Chemistry, University of Luebeck, Center for Structural and Cell Biology in Medicine, Ratzeburger Allee 160, D-23538 Luebeck, Germany.
authors.forename: Bärbel S
authors.initials: BS
authors.lastname: Blaum
authors.forename: Winfried
authors.initials: W
authors.lastname: Wünsche
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Benie
authors.forename: Yuri
authors.initials: Y
authors.lastname: Kusov
authors.forename: Hannelore
authors.initials: H
authors.lastname: Peters
authors.forename: Verena
authors.initials: V
authors.lastname: Gauss-Müller
authors.forename: Thomas
authors.initials: T
authors.lastname: Peters
authors.forename: Georg
authors.initials: G
authors.lastname: Sczakiel
mesh_terms: D000086042:3C Viral Proteases; D000998:Antiviral Agents / Q000737:chemistry; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D054856:G-Quadruplexes; D030041:Hepatitis A virus / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D012367:RNA, Viral / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D003854:Deoxyribonucleotides; D012367:RNA, Viral; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases