Wu Yuqin, Yang Qianfan, Wang Guan Alex, Li Feng
Key Laboratory of Green Chemistry and Technology of Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, Sichuan, China.
JACS Au. 2025 Jul 11;5(7):3632-3638. doi: 10.1021/jacsau.5c00689. eCollection 2025 Jul 28.
Folding and binding energies (Δ and Δ ) are critical thermodynamic parameters for characterizing the stability and functionality of aptamers. However, it remains difficult to directly characterize aptamer folding and binding in the native environment. Here, we describe a free energy shift assay (FESA) as a simple and instrument-free method to characterize the folding and binding thermodynamics of aptamers. FESA leverages the thermodynamic impact of the aptamer on a panel of toehold-mediated exchange reactions with well-characterized energetics to determine its folding and binding free energies. Using this principle, we successfully determined the Δ and Δ of a classic aptamer, thrombin binding aptamer (TBA), against varying cations, molecular crowding, and a cell-mimicking environment. Because our method is a molecular tool that can be easily adapted in any biochemistry laboratory without the need for specialized instruments, we anticipate that it will find broad applications in the thermodynamic characterization of aptamers and other functional or noncanonical nucleic acids.
折叠能和结合能（Δ 和Δ ）是用于表征适体稳定性和功能的关键热力学参数。然而，在天然环境中直接表征适体的折叠和结合仍然很困难。在此，我们描述了一种自由能位移测定法（FESA），作为一种简单且无需仪器的方法来表征适体的折叠和结合热力学。FESA利用适体对一组具有明确能量学特征的引发链介导的交换反应的热力学影响，来确定其折叠自由能和结合自由能。利用这一原理，我们成功测定了一种经典适体——凝血酶结合适体（TBA）在不同阳离子、分子拥挤环境和细胞模拟环境下的Δ 和Δ 。由于我们的方法是一种分子工具，无需专门仪器即可在任何生物化学实验室轻松应用，我们预计它将在适体以及其他功能性或非经典核酸的热力学表征中得到广泛应用。
