<br>鸟嘌呤、黄嘌呤、次黄嘌呤和腺嘌呤适体的亲和引导协同进化,ACS Chemical Biology

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鸟嘌呤、黄嘌呤、次黄嘌呤和腺嘌呤适体的亲和引导协同进化
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2024-01-09 , DOI: 10.1021/acschembio.3c00660
Yuzhe Ding ₁ , Lide Gu ₁ , Xiaoqin Wang ₁ , Ziyu Zhang ₁ , Hanxiao Zhang ₁ , Juewen Liu ₁

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多个适体的同时进化可以极大地提高适体发现的速度。以往的研究大多针对不同靶点使用相同的浓度，导致文库被一种或几种适体占据主导地位，成功率较低。为了培养最好的适体在序列空间中独立生长，重要的是（1）使用接近其解离常数的低靶浓度，以及（2）在任何序列开始占主导地位之前在早期一轮停止。在本研究中，我们展示了这种亲和力引导的选择概念，使用 capture-SELEX 方法分离四种重要嘌呤的适体：鸟嘌呤 (5 μM)、黄嘌呤 (50 μM)、次黄嘌呤 (10 μM) 和腺嘌呤 (10 μM) 。第 9 轮文库被拆分，在第 10 轮中，分别使用四个靶标来洗脱结合序列。利用硫黄素 T 荧光光谱和等温滴定量热法，我们确认了黄嘌呤、鸟嘌呤和腺嘌呤的高选择性适配体。这些适体的Kd值低于 1 μM，对大多数竞争性分析物的选择性约为 100 倍，与现有的 RNA 适体和核糖开关相比_，它们具有优势。单独使用次黄嘌呤进行单独选择，即使负选择也没有获得选择性适体，这解释了我们的混合选择中缺乏其适体。这项亲和引导的多重 SELEX 研究提供了适体选择的基本见解，并为三种重要的嘌呤提供了高质量的适体。

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Affinity-Guided Coevolution of Aptamers for Guanine, Xanthine, Hypoxanthine, and Adenine

The simultaneous evolution of multiple aptamers can drastically increase the speed of aptamer discovery. Most previous studies used the same concentration for different targets, leading to the dominance of the libraries by one or a few aptamers and a low success rate. To foster the best aptamers to grow independently in the sequence space, it is important to (1) use low target concentrations close to their dissociation constants and (2) stop at an early round before any sequence starts to dominate. In this study, we demonstrate this affinity-guided selection concept using the capture-SELEX method to isolate aptamers for four important purines: guanine (5 μM), xanthine (50 μM), hypoxanthine (10 μM), and adenine (10 μM). The round 9 library was split, and in round 10, the four targets were individually used to elute the binding sequences. Using thioflavin T fluorescence spectroscopy and isothermal titration calorimetry, we confirmed highly selective aptamers for xanthine, guanine, and adenine. These aptamers have K_d values below 1 μM and around 100-fold selectivity against most competing analytes, and they compare favorably with existing RNA aptamers and riboswitches. A separate selection was performed using hypoxanthine alone, and no selective aptamer was achieved, even with negative selection, explaining the lack of its aptamer in our mixed selection. This affinity-guided multiplex SELEX study offers fundamental insights into aptamer selection and provides high-quality aptamers for three important purines.

更新日期：2024-01-09

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