Structural characterization of an anti-gp120 RNA aptamer that neutralizes R5 strains of HIV-1
Structural characterization of an anti-gp120 RNA aptamer that neutralizes R5 strains of HIV-1
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作者：
Dey, AK;Griffiths, C;James, W
我们最近描述了2-氟嘧啶取代的RNA适体的分离，其特异性结合R5株HIV-1（Ba-L）的表面糖蛋白（gp 120），如先前的研究所示。这些适体有效地中和组织培养实验室适应株和来自多个进化枝的不同R5临床分离株的人外周血单核细胞中的HIV-1感染性。在这里，我们报告了一个这样的中和适体，B40，使用酶和化学探测方法的详细结构表征。我们确定的最小区域的适体结合gp 120必不可少的，并相应地设计了一个77个核苷酸的截短的适体，B40 t77。然后，我们定量分析亲本和截短的（最小）适体的结合亲和力和中和效力，并显示它们是相当的。此外，使用二级结构分析，RNA诱变和BIAcore表面等离子体共振（SPR）结合试验的结果，我们假设，折叠的RNA结构是需要提出特定的核苷酸序列，以允许gp 120的识别和结合。从这项研究中获得的信息可能为开发新的抗HIV-1疗法提供线索，并可用于扩展我们对病毒与其宿主细胞之间分子相互作用的理解。
We recently described the isolation of 2-fluoropyrimidine-substituted RNA aptamers that bind specifically to the surface glycoprotein (gp120) of the R5 strain, HIV-1(Ba-L), as presented in a previous study. These aptamers potently neutralize HIV-1 infectivity in human peripheral blood mononuclear cells of both tissue culture lab-adapted strains and diverse R5 clinical isolates from multiple clades. Here, we report a detailed structural characterization of one such neutralizing aptamer, B40, using enzymatic and chemical probing methods. We identify the minimal region of the aptamer essential for binding gp120 and accordingly design a 77-nucleotide truncated aptamer, B40t77. We then quantitatively analyze the binding affinity and neutralization potency of the parental and truncated (minimal) aptamer, and show them to be comparable. Furthermore, using results from secondary structure analysis, RNA mutagenesis and BIAcore surface plasmon resonance (SPR) binding assays, we hypothesize that a folded RNA structure is required to present specific nucleotide sequences to allow gp120-recognition and binding. The information gained from this study may provide leads for development of novel anti-HIV-1 therapies and can be used to extend our understanding of the molecular interactions between the virus and its host cell.