Institute of Advanced Energy, Kyoto University, Kyoto 611-0011, Japan.
Bioorg Med Chem. 2011 Aug 1;19(15):4473-81. doi: 10.1016/j.bmc.2011.06.031. Epub 2011 Jun 16.
A facile strategy of stepwise molding of a ribonucleopeptide (RNP) complex affords fluorescent RNP sensors with selective dopamine recognition. In vitro selection of a RNA-derived RNP library, a complex of the Rev peptide and its binding site Rev Responsive Element (RRE) RNA appended with random nucleotides in variable lengths, afforded RNP receptors specific for dopamine. The modular structure of the RNP receptor enables conversion of dopamine-binding RNP receptors to fluorescent dopamine sensors. Application of conditional selection schemes, such as the variation of salt concentrations and application of a counter-selection step by using a competitor ligand norepinephrine resulted in isolation of RNP receptors with defined dopamine-binding characteristics. Increasing the salt condition at the in vitro selection stage afforded RNP receptors with higher dopamine affinity, while addition of norepinephrine in the in vitro selection milieu at the counter-selection step reinforced the selectivity of RNP receptors to dopamine against norepinephrine. Thermodynamic analyses and circular dichroismic studies of the dopamine-RNP complexes suggest that the dopamine-binding RNP with higher selectivity against norepinephrine forms a pre-organized binding pocket and that the dopamine-binding RNP with higher affinity binds dopamine through the induced-fit mechanism. These results indicate that the selection condition controls the ligand-binding mechanism of RNP receptors.
一种逐步成型的核糖核肽（RNP）复合物的简便策略，提供了具有选择性多巴胺识别功能的荧光 RNP 传感器。通过体外选择 RNA 衍生的 RNP 文库，得到了一种 Rev 肽与其结合位点 Rev 反应元件（RRE）RNA 的复合物，其中随机核苷酸以不同的长度附加。这种复合物是具有多巴胺特异性的 RNP 受体。RNP 受体的模块化结构使得多巴胺结合的 RNP 受体能够转化为荧光多巴胺传感器。应用条件选择方案，如盐浓度的变化和使用竞争配体去甲肾上腺素进行反选择步骤，分离出具有特定多巴胺结合特性的 RNP 受体。在体外选择阶段增加盐条件可获得对多巴胺具有更高亲和力的 RNP 受体，而在反选择步骤中在体外选择环境中添加去甲肾上腺素可增强 RNP 受体对多巴胺与去甲肾上腺素的选择性。对多巴胺-RNP 复合物的热力学分析和圆二色性研究表明，对去甲肾上腺素具有更高选择性的多巴胺结合 RNP 形成了预组织的结合口袋，而对多巴胺具有更高亲和力的结合 RNP 通过诱导契合机制结合多巴胺。这些结果表明，选择条件控制 RNP 受体的配体结合机制。
