Templating effect in DNA proximity ligation enables use of non-bioorthogonal chemistry in biological fluids.

Nicholas G Spiropulos, Jennifer M Heemstra
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引用次数: 10

Abstract

Here we describe the first example of selective reductive amination in biological fluids using split aptamer proximity ligation (StAPL). Utilizing the cocaine split aptamer, we demonstrate small-molecule-dependent ligation that is dose-dependent over a wide range of target concentrations in buffer, human blood serum and artificial urine medium. We explore the substrate binding preferences of the split aptamer and find that the cinchona alkaloids quinine and quinidine bind to the aptamer with higher affinity than cocaine. This increased affinity leads to improved detection limits for these small-molecule targets. We also demonstrate that linker length and hydrophobicity impact the efficiency of split aptamer ligation. The ability to carry out selective chemical transformations using non-bioorthogonal chemistry in media where competing reactive groups are present highlights the power of the increased effective molarity provided by DNA assembly. Obviating the need for bioorthogonal chemistry would dramatically expand the repertoire of chemical transformations available for use in templated reactions such as proximity ligation assays, in turn enabling the development of novel methods for biomolecule detection.

DNA接近结扎中的模板效应使得在生物流体中使用非生物正交化学成为可能。
在这里,我们描述了生物流体中使用分裂适体接近结扎(StAPL)的选择性还原性胺化的第一个例子。利用可卡因分裂适体,我们证明了小分子依赖性结扎在缓冲液、人血清和人工尿液培养基中的靶浓度范围内是剂量依赖性的。我们探索了分裂适配体的底物结合偏好,发现金鸡纳生物碱奎宁和奎尼丁与适配体的结合比可卡因具有更高的亲和力。这种增加的亲和力导致这些小分子靶标的检测限提高。我们还证明了连接体长度和疏水性影响分裂适体连接的效率。在存在竞争性反应基团的介质中使用非生物正交化学进行选择性化学转化的能力突出了DNA组装提供的有效摩尔浓度增加的力量。消除对生物正交化学的需求将极大地扩大可用于模板反应的化学转化的范围,例如接近连接测定,从而使生物分子检测的新方法得以发展。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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