Institute of Chemistry, University of Luebeck, Center for Structural and Cell Biology in Medicine, Ratzeburger Allee 160, D-23538 Luebeck, Germany.
Nucleic Acids Res. 2012 Apr;40(7):3042-55. doi: 10.1093/nar/gkr1152. Epub 2011 Dec 10.
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.
长度短至 6 个核苷酸的寡核苷酸已被证明可以特异性和紧密地与蛋白质结合，并影响其生物学功能。然而，相应复合物的结构数据很少。我们使用最近开发的六核苷酸阵列，鉴定出与甲型肝炎病毒 (HAV 3C(pro)) 的 3C 蛋白酶特异性结合的六脱氧核苷酸。体外抑制实验鉴定出六核苷酸 5'-GGGGGT-3' (G(5)T) 是 3C(pro) 蛋白酶抑制剂。通过 (1)H NMR 光谱，发现 G(5)T 形成 G-四链体，这可能被认为是最小的适体。借助 (1)H、(15)N-HSQC 实验，确定了 G(5)T 的结合位点位于 HAV 3C(pro) 的 C 末端 β-桶。重要的是，以前被鉴定为潜在病毒 RNA 结合位点的高度保守的 KFRDI 基序不是 G(5)T 结合位点的一部分，并且 G(5)T 也不干扰病毒 RNA 的结合。我们的研究结果表明，寡核苷酸与蛋白质的序列特异性相互作用发生在短至六核苷酸的寡核苷酸上，这表明这些化合物可能具有药物相关性。
