High-Throughput Strategy for Enhancing Aptamer Performance across Different Environmental Conditions.
High-Throughput Strategy for Enhancing Aptamer Performance across Different Environmental Conditions.
复制标题
增强适配体在不同环境条件下性能的高通量策略。
DOI：
10.1021/acssensors.2c02106
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发表时间：
2023
期刊：
影响因子：
8.9
通讯作者：
Soh,HTom
中科院分区：
文献类型：
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作者：
Wan,Leighton;Yoshikawa,Alex;Eisenstein,Michael;Soh,HTom
在特定环境条件下选择的适体（例如，pH、离子浓度和温度）在用于其他环境时通常表现出大大降低的亲和力。这对于生物医学应用尤其成问题，其中适体暴露于具有独特化学性质的样品基质，例如血液、汗液或尿液。我们提出了一个高通量筛选程序，以适应现有的适体中使用的样品，其化学组成与原来的选择条件有很大的不同。在我们小组先前工作的基础上，我们利用了一种改良的DNA测序仪，能够在所需的测定条件下筛选多达107个独特的适体突变体与靶结合。作为一个例子，我们筛选了所有11，628个单取代和双取代突变体，用于先前报道的葡萄糖适体，该适体最初在高离子强度缓冲液中选择，并在生理条件下表现出相对较低的亲和力。在单轮筛选后，我们鉴定了在生理条件下亲和力增加了100倍的适体突变体。有趣的是，我们发现单碱基取代的影响相对温和，但在双突变体中观察到相当大的结合改善，突出了突变之间合作效应的重要性。这种方法应该推广到其他适体和环境条件的一系列应用。
Aptamers selected under specific environmental conditions (e.g., pH, ion concentration, and temperature) often exhibit greatly reduced affinity when used in other contexts. This can be especially problematic for biomedical applications in which aptamers are exposed to sample matrices with distinctive chemical properties, such as blood, sweat, or urine. We present a high-throughput screening procedure for adapting existing aptamers for use in samples whose chemical composition differs considerably from the original selection conditions. Building on prior work from our group, we have utilized a modified DNA sequencer capable of screening up to 107unique aptamer mutants for target binding under the desired assay conditions. As an exemplar, we screened all 11,628 single- and double-substitution mutants for a previously reported glucose aptamer that was originally selected in high-ionic strength buffer and exhibited relatively low affinity in physiological conditions. After a single round of screening, we identified aptamer mutants with ∼four-fold increased affinity in physiological conditions. Interestingly, we found that the impact of single-base substitutions was relatively modest but observed considerably greater binding improvements among the double mutants, highlighting the importance of cooperative effects between mutations. This approach should be generalizable to other aptamers and environmental conditions for a range of applications.