CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China , Hefei, Anhui 230026, P. R. China.
Anal Chem. 2014 Jun 3;86(11):5559-66. doi: 10.1021/ac501061c. Epub 2014 May 16.
Although much effort has been made for studies on aptamer-target interactions due to promising applications of aptamers in biomedical and analytical fields, measurement of the aptamer-target binding constant and binding site still remains challenging. Herein, we report a sensitive label-free chemiluminescence (CL) strategy to determine the target concentration and, more importantly, to measure the target-aptamer binding constant and binding site. This approach is suitable for multiple types of targets, including small molecules, peptides, and proteins that can enhance the CL initiated by N-(aminobutyl)-N-ethylisoluminol functionalized gold colloids, making the present method a general platform to investigate aptamer-target interactions. This approach can achieve extremely high sensitivity with nanogram samples for measuring the target-aptamer binding constant. And the measurement could be rapidly performed using a simple and low-cost CL system. It provides an effective tool for studying the binding of biologically important molecules to nucleic acids and the selection of aptamers. Besides, we have also discovered that the 14-mer aptamer fragment itself split from the ATP-binding aptamer could selectively capture ATP. The binding constant, site, and conformation between ATP and the 14-mer aptamer fragment were obtained using such a novel CL strategy and molecular dynamic simulation.
尽管由于适体在生物医学和分析领域的应用前景，人们已经为适体-靶标相互作用的研究做出了很大的努力，但适体-靶标结合常数和结合位的测量仍然具有挑战性。在此，我们报告了一种灵敏的无标记化学发光（CL）策略，用于测定靶标浓度，更重要的是，用于测量靶-适体结合常数和结合位。该方法适用于多种类型的靶标，包括小分子、肽和蛋白质，它们可以增强 N-(氨丁基)-N-乙基异鲁米诺功能化金胶体引发的 CL，使本方法成为研究适体-靶标相互作用的通用平台。该方法可以用纳克级的样品实现极高的灵敏度来测量靶-适体结合常数。并且可以使用简单且低成本的 CL 系统快速进行测量。它为研究生物重要分子与核酸的结合以及适体的选择提供了有效的工具。此外，我们还发现，从 ATP 结合适体中分离出来的 14 -mer 适体片段本身可以选择性地捕获 ATP。使用这种新型 CL 策略和分子动力学模拟获得了 ATP 与 14-mer 适体片段之间的结合常数、结合位和构象。
