A system for multiplexed selection of aptamers with exquisite specificity without counterselection.
A system for multiplexed selection of aptamers with exquisite specificity without counterselection.
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DOI：
10.1073/pnas.2119945119
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发表时间：
2022-03-22
影响因子：
11.1
通讯作者：
Soh HT
中科院分区：
文献类型：
--
作者：
Yoshikawa AM;Wan L;Zheng L;Eisenstein M;Soh HT
适配体具有区分结构相似的小分子的能力。然而，使用基于对非目标分子的反选择的传统方法来产生这种高度特异性的适体已被证明是具有挑战性的。在这项工作中，我们描述了一个高通量筛选平台，该平台可以在单个实验中表征数百万个适体对一组结构相关分子的特异性，并在没有任何反选择的情况下生成精细特异性的适体。作为范例，我们利用我们的平台生成了对三种结构相关的犬尿氨酸代谢物具有高亲和力和特异性的适配体。我们的平台可以很容易地适应其他小分子靶标，因此应该加速适体试剂的开发，具有精致的特异性。适体已被证明是检测小分子的有价值的工具，因为它们具有非凡的能力，可以特异性地区分结构相似的分子。大多数适体选择工作都依赖于反选择来消除对干扰物或与感兴趣的目标结构相似的亲属表现出不必要的交叉反应性的适体。然而，由于适体库的亲和性和特异性特征基本上是不可知的，因此不可能确定最佳的反选择参数。因此，反选择实验需要尝试和错误的方法，这种方法本质上是低效的，并且可能无法产生具有亲和力和特异性最佳组合的适体。在这项工作中，我们描述了一种高通量筛选过程，用于并行生成针对多个目标的高特异性适配体，同时也消除了反选择的需要。我们采用基于改进台式测序仪的平台进行大规模平行适配体筛选过程，从而在单个实验中针对多个结构相似的分子选择高度特异性的适配体，而无需任何反选择。作为证明，我们在一次选择实验中为三种结构相似的犬尿氨酸代谢物选择了具有高亲和力和精致特异性的适配体，这些适配体只存在一个羟基的差异。这一过程可以很容易地适用于其他小分子分析物，并将大大加快适体试剂的开发，使其对目标分析物具有精细的特异性。
Aptamers have the capacity to discriminate between structurally similar small molecules. However, generating such highly specific aptamers has proven challenging using conventional processes based on counterselection against nontarget molecules. In this work, we describe a high-throughput screening platform that can characterize the specificity of millions of aptamers toward a group of structurally related molecules in a single experiment and generate exquisitely specific aptamers without any counterselection. As exemplars, we generated aptamers with high affinity and specificity toward three structurally related kynurenine metabolites using our platform. Our platform can be readily adapted to other small-molecule targets and should therefore accelerate the development of aptamer reagents with exquisite specificity. Aptamers have proven to be valuable tools for the detection of small molecules due to their remarkable ability to specifically discriminate between structurally similar molecules. Most aptamer selection efforts have relied on counterselection to eliminate aptamers that exhibit unwanted cross-reactivity to interferents or structurally similar relatives to the target of interest. However, because the affinity and specificity characteristics of an aptamer library are fundamentally unknowable a priori, it is not possible to determine the optimal counterselection parameters. As a result, counterselection experiments require trial-and-error approaches that are inherently inefficient and may not result in aptamers with the best combination of affinity and specificity. In this work, we describe a high-throughput screening process for generating high-specificity aptamers to multiple targets in parallel while also eliminating the need for counterselection. We employ a platform based on a modified benchtop sequencer to conduct a massively parallel aptamer screening process that enables the selection of highly specific aptamers against multiple structurally similar molecules in a single experiment, without any counterselection. As a demonstration, we have selected aptamers with high affinity and exquisite specificity for three structurally similar kynurenine metabolites that differ by a single hydroxyl group in a single selection experiment. This process can easily be adapted to other small-molecule analytes and should greatly accelerate the development of aptamer reagents that achieve exquisite specificity for their target analytes.
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DOI：
10.1073/pnas.1504354112
发表时间：
2015-05-26
影响因子：
11.1
作者：
You, Mingxu;Litke, Jacob L.;Jaffrey, Samie R.
通讯作者：
Jaffrey, Samie R.
与 L-色氨酸结合的 DNA 适体的表征和应用
影响因子：
4.2
作者：
Yang, Xiaojuan;Bing, Tao;Shangguan, Dihua
通讯作者：
Shangguan, Dihua
DOI：
10.1126/science.1218298
发表时间：
2012-03-09
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
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通讯作者：
Jaffrey SR
影响因子：
11.4
作者：
HARADA, K;FRANKEL, AD
通讯作者：
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影响因子：
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作者：
Stoltenburg R;Nikolaus N;Strehlitz B
通讯作者：
Strehlitz B