Beacons Contribute Valuable Empirical Information to Theoretical 3-D Aptamer-Peptide Binding
Beacons Contribute Valuable Empirical Information to Theoretical 3-D Aptamer-Peptide Binding
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DOI：
10.1007/s10895-019-02380-6
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发表时间：
2019-05-01
影响因子：
2.7
通讯作者：
Phillips, Taylor
中科院分区：
文献类型：
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作者：
Bruno, John G.;Phillips, Taylor
根据抗巨细胞病毒和抗单纯疱疹病毒-2抗体的已知结合区的5个不同的多肽，开发了DNA适配子，并基于ELASA微板法对其进行了相对亲和力排序。研究了前五个亲和力最高的适配子的二级结构，以寻找茎环的共性和最可能的肽结合位点。通过在5‘端添加Tye 665染料和在3’端添加爱荷华州黑色猝灭剂，这些茎-环结构中的两个被转化为信标。当与浓度增加的五种多肽竞争时，可能的十种相互作用中只有三种显示了荧光信标反应。当通过PDB文件的生成进行建模时，在通过PATCHDOCK和Yasara后，两个适配子信标-肽的相互作用没有显示出分离G-C茎环区域的理论证据，尽管明确的经验证据表明，荧光团和猝灭剂在Forster距离之外分离导致了丰富的荧光。在第二个信标的例子中，Yasara模型表明信标始终是开放的，尽管有明确的经验证据表明它不是(没有荧光反应)，并且只有在五种多肽中的一种存在时才打开。这些结果被解释为一个证据，即基于刚性受体-配体形状互补的三维对接软件，如PATCHDOCK和Yasara，可能不能反映适体与其同源靶标之间的诱导匹配作用。因此，为了获得最完整和准确的适配子-肽结合的图像，可能需要几种理论和经验(例如信标荧光)分析方法。
DNA aptamers were developed against five different peptides from the known binding regions of anti-Cytomegalovirus and anti-Herpes Simplex Virus-2 antibodies and the aptamers were ranked by relative affinity based on an ELISA-like (ELASA) microplate assay. The secondary structures of the top five highest affinity aptamers were studied for stem-loop commonalities and the most probable peptide binding sites. Two of these stem-loop structures were converted into beacons by addition of TYE 665 dye on the 5 ' end and Iowa Black quencher on the 3 ' end. When competed against increasing concentrations of each of the five peptides, only three of the possible ten interactions demonstrated lights on fluorescence beacon responses. When modeled by generation of PDB files, after passage through PATCHDOCK and YASARA, two of the aptamer beacon-peptide interactions showed no theoretical evidence of separating the G-C stem-loop region, despite clear empirical evidence of separation of the fluorophore and quencher beyond the Forster distance leading to abundant fluorescence. And in the second beacon's case, YASARA modeling suggested that the beacon was always open despite clear empirical evidence that it was not (no fluorescence response) and only opened in the presence of one of the five peptides. These results are interpreted as a demonstration that 3-dimensional docking software such as PATCHDOCK and YASARA, which are based on rigid receptor-ligand shape complementarity may not reflect the induced-fit interactions between aptamers and their cognate targets. Therefore, for the most complete and accurate picture of aptamer-peptide binding, several theoretical and empirical (e.g., beacon fluorescence) analysis methods may be needed.