Aptamers can discriminate alkaline proteins with high specificity.
Aptamers can discriminate alkaline proteins with high specificity.
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DOI：
10.1002/cbic.201100252
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发表时间：
2011-11-25
期刊：
影响因子：
3.2
通讯作者：
Chaput, John C.
中科院分区：
文献类型：
--
作者：
Yu, Hanyang;Jiang, Bing;Chaput, John C.
适配子是单链核酸，可以折叠成稳定的三维结构，其配体结合部位在形状上是互补的，并与所需的靶标荷电。适配子是通过一种称为体外选择的迭代过程产生的，这使得它们可以从随机序列池中分离出来。虽然适配子已经被选择来结合广泛的靶标，但人们普遍认为，由于控制相反电荷聚合物的吸引力(例如，聚电解质效应)，这些分子无法区分强碱性蛋白质。组蛋白是组成核小体核心结构的真核蛋白，是研究适配子结合特性的有趣靶点，因为这些蛋白具有带正电的表面，通过非共价序列无关的相互作用与DNA结合。我们实验室和其他实验室之前的选择已经产生了对单个组蛋白具有高亲和力但低特异性的DNA适配子。这是否是适配子的普遍限制是一个有趣的问题，对蛋白质亲和试剂的未来发展具有重要的实际意义。在这里，我们报道了体外筛选的DNA适配子，它与组蛋白H4结合，Kd值为13 nM，并与其他核心组蛋白区分100-480倍，相当于高达3.4kcal/ΔΔ的G值。这一结果扩展了我们对适配子的基本理解，包括折叠成选择性结合碱性蛋白的形状的能力。
Aptamers are single-stranded nucleic acids that fold into stable three-dimensional structures with ligand binding sites that are complementary in shape and charge to a desired target. Aptamers are generated by an iterative process known as in vitro selection, which permits their isolation from pools of random sequences. While aptamers have been selected to bind a wide range of targets, it is generally thought that these molecules are incapable of discriminating strongly alkaline proteins due to the attractive forces that govern oppositely charged polymers (e.g., polyelectrolyte effect). Histones, eukaryotic proteins that make up the core structure of nucleosomes are interesting targets for exploring the binding properties of aptamers because these proteins have positively charged surfaces that bind DNA through non-covalent sequence-independent interactions. Previous selections by our lab and others have yielded DNA aptamers with high affinity but low specificity to individual histone proteins. Whether this is a general limitation of aptamers is an interesting question with important practical implications in the future development of protein affinity reagents. Here we report the in vitro selection of a DNA aptamer that binds to histone H4 with a Kd of 13 nM and distinguishes other core histone proteins by 100 to 480-fold, which corresponds to a ΔΔG of up to 3.4 kcal/mol. This result extends our fundamental understanding of aptamers to include the ability to fold into shapes that selectively bind alkaline proteins.