Qu Hao, Zheng Manyi, Ma Qihui, Wang Lu, Mao Yu, Eisenstein Michael, Tom Soh Hyongsok, Zheng Lei
School of Food and Biological Engineering and Engineering Research Center of Bioprocess of Ministry of Education, Hefei University of Technology, Hefei, 230009, China.
Department of Electrical Engineering and Department of Radiology, Stanford University, Stanford, CA 94305, USA.
Angew Chem Int Ed Engl. 2023 Mar 1;62(10):e202214045. doi: 10.1002/anie.202214045. Epub 2023 Jan 31.
The capacity to precisely modulate aptamer affinity is important for a wide variety of applications. However, most such engineering strategies entail laborious trial-and-error testing or require prior knowledge of an aptamer's structure and ligand-binding domain. We describe here a simple and generalizable strategy for allosteric modulation of aptamer affinity by employing a double-stranded molecular clamp that destabilizes aptamer secondary structure through mechanical tension. We demonstrate the effectiveness of the approach with a thrombin-binding aptamer and show that we can alter its affinity by as much as 65-fold. We also show that this modulation can be rendered reversible by introducing a restriction enzyme cleavage site into the molecular clamp domain and describe a design strategy for achieving even more finely-tuned affinity modulation. This strategy requires no prior knowledge of the aptamer's structure and binding mechanism and should thus be generalizable across aptamers.
精确调节适体亲和力的能力对于广泛的应用来说至关重要。然而，大多数此类工程策略需要进行费力的反复试验测试，或者需要预先了解适体的结构和配体结合域。我们在此描述一种简单且可推广的策略，通过使用双链分子钳来变构调节适体亲和力，该分子钳通过机械张力破坏适体二级结构。我们用凝血酶结合适体证明了该方法的有效性，并表明我们可以将其亲和力改变多达65倍。我们还表明，通过在分子钳结构域引入限制性内切酶切割位点，这种调节可以变为可逆的，并描述了一种实现更精细亲和力调节的设计策略。该策略不需要预先了解适体的结构和结合机制，因此应该可以推广到各种适体。
