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

核酸适体是一种短的核酸结合物种，由于其结合大量配体（包括氨基酸、含核糖的辅因子和药物）的显著能力而成为分子识别事件的有希望的候选者。这些适体对靶配体的高亲和力和特异性通常通过分子形状互补性、氢键和堆叠相互作用的组合来实现。此类结合事件通常涉及适体的配体诱导的结构变化，导致形成负责配体结合的独特二级结构，其中内部或茎环结构代表结合事件的活性位点。然而，对于小配体的RNA和DNA适体的进一步设计，很少关注其他类型的非碱基配对部分，尽管它们具有结合口袋的潜力。 关于我们 所述双链体中的脱碱基（AP）位点被用作结合生物学上重要的配体的活性腔，其中所述双链体中的脱碱基（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，强烈预期通过与AP位点侧翼的两个核苷酸堆叠，靶配体插入AP位点，并且这伴随着与AP位点相对的受体核苷酸形成氢键。实际上，我们在本研究项目中已经成功地开发了这类对核黄素（解离常数Kd =1.9μM）、茶碱（Kd =31μM）、咖啡因（Kd =20μM）和腺苷（检测范围：10~150μM）具有高选择性的适配子。与典型的RNA适体相比，我们的基于AP位点的DNA适体具有一些优点，例如它们的容易和低成本合成，以及更高的化学稳定性。这些特征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