Base-Pairing at the AbasicSite in DNA Duplexes and Its Application for Adenosine Aptasensors
Base-Pairing at the AbasicSite in DNA Duplexes and Its Application for Adenosine Aptasensors
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DNA 双链体中脱碱基位点的碱基配对及其在腺苷适体传感器中的应用
DOI：
10.1002/cbic.201100666
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发表时间：
2012
期刊：
影响因子：
3.2
通讯作者：
S. Nishizawa and N. Teramae
中科院分区：
文献类型：
--
作者：
Y. Pang;Z. Xu;Y. Sato;S. Nishizawa and N. Teramae
通过热变性和等温滴定量热（ITC）实验研究了核苷与碱性位点（AP位点）-含有与AP位点相反的互补核苷的DNA双链（AP - DNA）的结合。嘌呤核苷与AP - DNA的结合具有很高的亲和力（对腺苷来说Kd=14.1 μ m，对鸟苷来说Kd= 41.8 μ m），这种相互作用主要是由焓变驱动的，类似于DNA插入物的情况。相比之下，嘧啶核苷与AP‐dna的结合并不明显，这表明AP位点的堆叠相互作用在嘌呤核苷与AP‐dna的结合中起着关键作用。接下来，应用AP - DNA作为腺苷的适配体，在腺苷和AP -位点结合荧光配体之间进行竞争分析。该试验采用荧光配体核黄素，它与DNA双链中的AP位点结合，从而引起荧光猝灭。通过将腺苷加入到核黄素/AP‐DNA复合体中，腺苷与AP位点的结合导致核黄素从AP位点释放，从而导致核黄素荧光的恢复。AP‐dna可以作为一类新的适体传感器，对腺苷的检出限为0.7 μ m。与传统的腺苷适配体传感器相比，本方法对腺苷的选择性高于其他核苷酸（AMP， ADP和ATP）。该方法不需要对荧光团进行共价标记，因此具有成本效益；结果表明，该方法适用于马血清中腺苷的检测。
The binding of nucleosides to abasic site (AP site)‐containing DNA duplexes (AP‐DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (Kd=14.1 μMfor adenosine and 41.8 μMfor guanosine) for binding to the AP‐DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP‐DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP‐DNAs, as revealed by ITC measurements. Next, to apply an AP‐DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP‐site‐binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP‐DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP‐DNAs can serve as a new class of aptasensors—a limit of detection of 0.7 μMwas obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost‐effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.