Functional binding of hexanucleotides to 3C protease of hepatitis A virus

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′ (G₅T) as a 3C^pro protease inhibitor. Using ¹H NMR spectroscopy, G₅T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of ¹H, ¹⁵N-HSQC experiments the binding site for G₅T 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 G₅T-binding site, nor does G₅T 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.