In vitro evolution of functional DNA using capillary electrophoresis
In vitro evolution of functional DNA using capillary electrophoresis
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DOI：
10.1021/ja037832s
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发表时间：
2004-01-14
影响因子：
15
通讯作者：
Bowser, MT
中科院分区：
文献类型：
--
作者：
Mendonsa, SD;Bowser, MT
毛细管电泳电泳法(CE)首次被用于使用SELEX(通过指数富集法的配体的系统进化)来分离功能核酸序列。SELEX利用分子进化从随机RNA或DNA文库中选择功能序列(适配子)。传统的SELEX通常使用亲和层析进行，这可能会在选择步骤中引入显著的偏差。重要的偏差包括洗脱强结合序列的缓慢动力学，以及在目标分子系在固定载体上而不是在自由溶液中进行选择。在这种新颖的CE-SELEX方法中，选择发生在自由溶液中。结合靶标的核酸序列经历了迁移率的变化，以不同的速度迁移，使它们与非活性序列分离。因此，不需要像传统的SELEX那样将活性序列从柱上洗去，消除了任何动力学偏差。在这项工作中，CE-SELEX通过对免疫球蛋白E(IgE)进行选择来证明其可行性。仅两轮筛选即可获得解离常数低至40 nM的抗IgE适配子。
Electrophoretic selection with capillary electrophoresis (CE) is used, for the first time, to isolate functional nucleic acid sequences using SELEX (systematic evolution of ligands by exponential enrichment). SELEX uses molecular evolution to select functional sequences (aptamers) from random RNA or DNA libraries. Conventional SELEX is usually performed with affinity chromatography, which may introduce significant bias into the selection step. Important biases include the slow kinetics involved in the elution of strongly bound sequences and performing the selection with the target molecule tethered to the stationary support, not in free solution. In this novel CE-SELEX approach, selection occurs in free solution. The nucleic acid sequences that bind the target undergo a mobility shift, migrating at a different rate, allowing them to be separated from the inactive sequences. Thus, there is no need to wash the active sequences off a column as in conventional SELEX, eliminating any kinetic bias. In this work, the viability of CE-SELEX was demonstrated by performing selections against immunoglobulin E (IgE). Anti-IgE aptamers with dissociation constants as low as 40 nM were obtained in only two rounds of selection.