Development of Abasic Site-Containing DNA Duplex Aptamers for Biologically Important Substrates

核酸适配体是一种短核酸结合物种，由于其结合大量配体（包括氨基酸、含核糖的辅因子和药物）的显著能力，已成为分子识别事件的有希望的候选者。这些适体对目标配体的高亲和力和特异性通常是通过分子形状互补、氢键和堆叠相互作用的结合来实现的。这种结合事件通常涉及配体诱导的适体结构变化，导致形成与配体结合有关的独特二级结构，其中内环或茎环结构是结合事件的活性位点的代表。然而，对于小配体的RNA和DNA适体的进一步设计，其他类型的非碱基配对部分尽管具有结合口袋的潜力，但很少受到关注。在这里，我们报道了一类新的DNA双工核酸适体，用于选择性和更强地结合生物重要的配体，其中双工中的一个碱基（AP）位点被用作结合生物重要配体的活性腔，其中双工中的一个碱基（AP）位点被用作结合事件的活性腔。对于基于AP位点的DNA适体，我们强烈期望通过与AP位点两侧的两个核苷酸堆叠，靶配体嵌入到AP位点，这伴随着与AP位点对面的受体核苷酸形成氢键。事实上，我们在本项目中已经成功开发出对核黄素（解离常数K_d=1.9μM）、茶碱（K_d=31μM）、咖啡因（K_d=20μM）和腺苷（检测范围10~150μM）具有高选择性的核酸适体。与典型的RNA适配体相比，我们的基于AP位点的DNA适配体具有合成简单、成本低、化学稳定性高等优点。这些特性将允许各种分析应用。少

Aptamers, short nucleic acid-binding species, have emerged as promising candidates for molecular recognition events on account of their significant ability to bind large number of ligands, including amino acids, ribose-containing cofactors and drugs. High affinity and specificity of these aptamers toward target ligands are generally achieved by a combination of molecular shape complementarity, hydrogen-bonding and stacking interactions. Such binding events typically involve the ligand-induced structural changes of the aptamers, resulting in the formation of unique secondary structures responsible to the ligand binding, for which the internal - or stem-loop structures are representative of active sites for binding events. However, for the further design of both RNA and DNA aptamers for small ligands, little attention has been paid to other types of non-base-pairing moieties despite the potential as binding pockets.Here we report on a new class of DNA duplex aptamers for selective and st … More rong binding to biologically important ligands, where an abasic (AP) site in the duplex is utilized as an active cavity for binding to biologically important ligands, where an abasic (AP) site in the duplex is utilized as an active cavity for binding events. For the AP site-based DNA aptamers, it is strongly expected that, by stacking with two nucleotides flanking the AP site, a target ligand is intercalated into the AP site, and this is accompanied by the formation of hydrogen bonds with a receptor nucleotide opposite the AP site. Indeed, we have successfully developed this type of aptamers with high selectivity for riboflavin (dissociation constant K_d=1.9μM), theophylline (K_d=31μM), caffeine (K_d=20μM), and adenosine (detection range: 10~150μM) in this research project. As compared to typical RNA aptamers, our AP site-based DNA aptamers have some advantages, such as their easy and low-cost synthesis, and higher chemical stability. These features would allow various kinds of analytical applications. Less