Università di Napoli Federico II, Via Cintia, Napoli, Italia.
Nucleic Acids Res. 2011 Sep 1;39(17):7858-67. doi: 10.1093/nar/gkr522. Epub 2011 Jun 28.
Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human α-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5'-5' polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin-mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin-aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.
适体是一种能够识别分子靶标并能作为直接蛋白质抑制剂发挥作用的结构寡核苷酸。最著名的例子是凝血酶结合适体（TBA），它是一种 15 个碱基的单链 DNA，能够抑制凝血酶（凝血级联反应的关键酶）的活性。TBA 形成 G-四链体结构，这一点已被其 NMR 结构所证明。TBA 与人 α-凝血酶复合物的 X 射线结构在 2.9 Å 分辨率下得到解决，但没有提供适体构象和与蛋白质分子相互作用的细节。TBA 易被核酸酶降解。为了改善 TBA 的性质，已经产生了许多修饰的类似物。特别是，一种含有 5'-5'极性反转位点的修饰 TBA（mTBA），相对于 TBA 具有更高的稳定性和对凝血酶的亲和力，尽管其抑制活性较低。我们提出了凝血酶-mTBA 复合物的晶体结构，分辨率为 2.15 Å；最终的模型提供了一个清晰的凝血酶-适体相互作用的图像，并突出了 TBA 和 mTBA 不同性质的结构基础。我们的发现为设计具有改进抗凝活性的改良适体开辟了道路。
