Ding Yuzhe, Zhang Ziyu, Liu Juewen
Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada.
Chembiochem. 2024 Dec 2;25(23):e202400570. doi: 10.1002/cbic.202400570. Epub 2024 Oct 27.
During an aptamer selection, using a lower target concentration may result in aptamers with a higher binding affinity. Consequently, this begs the question of whether there is a lower limit for target concentration. In this work, we conducted three aptamer selections using 5 μM, 500 nM and 50 nM guanine as the targets, respectively. Successful enrichment of the same guanine aptamers was achieved at both 5 μM and 500 nM guanine, but not with 50 nM. Using 5 μM guanine, the aptamer was enriched in eight rounds of selection, compared to that for 500 nM, which was accomplished in 17 rounds. We discuss the relation of optimal target concentration to the observed K value of the resulting aptamers, of which the highest affinity aptamer had a measured K of 200 nM. Additionally, we investigated the binding of the aptamers through mutation studies, revealing a critical cytosine. Mutating this cytosine to a thymine switched the selectivity from guanine to adenine, which is reminiscent of the guanine riboswitch. This study revealed a limit in using low target concentration, and the insights described in this article will be useful for guiding the choice of target concentration during capture-SELEX.
在适体筛选过程中，使用较低的靶标浓度可能会产生具有更高结合亲和力的适体。因此，这就引出了一个问题，即靶标浓度是否存在下限。在这项工作中，我们分别以5 μM、500 nM和50 nM鸟嘌呤作为靶标进行了三次适体筛选。在5 μM和500 nM鸟嘌呤浓度下均成功富集到了相同的鸟嘌呤适体，但在50 nM时未成功。使用5 μM鸟嘌呤时，适体在八轮筛选中得到富集，而使用500 nM鸟嘌呤时则在17轮筛选中才完成富集。我们讨论了最佳靶标浓度与所得适体观察到的K值之间的关系，其中亲和力最高的适体测得的K值为200 nM。此外，我们通过突变研究调查了适体的结合情况，发现了一个关键的胞嘧啶。将这个胞嘧啶突变为胸腺嘧啶会使选择性从鸟嘌呤转变为腺嘌呤，这让人联想到鸟嘌呤核糖开关。这项研究揭示了使用低靶标浓度的局限性，本文所述的见解将有助于指导捕获-SELEX过程中靶标浓度的选择。
