Mannironi C, Di Nardo A, Fruscoloni P, Tocchini-Valentini G P
Institute of Cell Biology, CNR, Rome, Italy.
Biochemistry. 1997 Aug 12;36(32):9726-34. doi: 10.1021/bi9700633.
RNA aptamers that specifically bind dopamine have been isolated by in vitro selection from a pool of 3.4 x 10(14) different RNA molecules. One aptamer (dopa2), which dominated the selected pool, has been characterized and binds to the dopamine affinity column with a dissociation constant of 2.8 microM. The specificity of binding has been determined by studying binding properties of a number of dopamine-related molecules, showing that the interaction with the RNA might be mediated by the hydroxyl group at position 3 and the proximal aliphatic chain in the dopamine molecule. The binding domain was initially localized by boundary experiments. Further definition of the dopamine binding site was obtained by secondary selection on a pool of sequences derived from a partial randomization of the dopa2 molecule. Sequence comparison of a large panel of selected variants revealed a structural consensus motif among the active aptamers. The dopamine binding pocket is built up by a tertiary interaction between two stem and loop motifs, creating a stable framework in which five invariant nucleotides are precisely arrayed. Minimal active sequence and key nucleotides have been confirmed by the design of small functional aptamers and mutational analysis. Enzymatic probing suggests that the RNA might undergo a conformational change upon ligand binding that stabilizes the proposed tertiary structure.
通过从3.4×10¹⁴个不同RNA分子的文库中进行体外筛选，已分离出特异性结合多巴胺的RNA适配体。在所选文库中占主导地位的一种适配体（dopa2）已被表征，它以2.8微摩尔的解离常数与多巴胺亲和柱结合。通过研究多种多巴胺相关分子的结合特性确定了结合的特异性，结果表明与RNA的相互作用可能由多巴胺分子中3位的羟基和近端脂肪链介导。结合结构域最初通过边界实验定位。通过对源自dopa2分子部分随机化的序列文库进行二次筛选，进一步明确了多巴胺结合位点。对大量选定变体的序列比较揭示了活性适配体之间的结构共有基序。多巴胺结合口袋由两个茎环基序之间的三级相互作用形成，形成了一个稳定的框架，其中五个不变核苷酸精确排列。通过设计小的功能性适配体和突变分析，证实了最小活性序列和关键核苷酸。酶促探测表明，RNA可能在配体结合时发生构象变化，从而稳定所提出的三级结构。
