靶标诱导变构解离SELEX技术筛选皮质醇特异核酸适体

Screening aptamers of cortisol by target-induced-structure-switching-dissociation SELEX

目的通过指数富集配体系统进化(SELEX)技术获得皮质醇特异性核酸适体,建立高品质、超敏、易用和稳定的皮质醇检测试剂盒。方法人工合成两端固定序列,中间为35 bp随机序列,生成80 bp长度ssDNA文库,捕获序列固定在磁珠上,文库与其退火组装,皮质醇的特异核酸适体通过我们建立的靶标诱导变构解离SELEX(TISSD-SELEX)技术获得。最后一轮的PCR产物经克隆测序,MegAlign和RNAstructure软件分析其一级和二级结构。实时(real-time)apta-PCR方法分析G12的亲和活性。结果经过12轮筛选后,获得10个皮质醇的核酸适体。序列分析显示,特异核酸适体以高G含量核酸序列为主,软件预测其中8个序列主要以G四联体二级结构为主,候选核酸适体G12显示了可以作为生物探针用于皮质醇检测。结论成功建立TISSD-SELEX技术,获得小分子靶标皮质醇核酸适体。小分子靶标无需将其偶联在固相介质或大分子上,对靶分子结构、性质无特殊要求,因而有望成为小分子靶标核酸适体筛选的通用技术方法。

ObjectiveTo obtain and identify aptamers which can specifically bind to cortisol by systematic evolution of ligands by exponential enrichment（SELEX） protocol,in order to establish a high-quality,ultra-sensitive,simple robust cortisol detection kit.MethodsA synthesized 80mer single stranded DNA random library containing 35 random sequences flanked by invariant primer was designed.Capture sequences were absorbed on the magnetic beads,the library and capture sequences were annealed and assembled,and the aptamers binding cortisol were obtained by the protocol which we had established as a screening method of target-induced-structure-switching-dissociation（TISSD）-SELEX.PCR products of the last round selection were cloned and sequenced.MegAlign and RNAstructure software was employed to analyze the primary structure and secondary structures of the aptamers.Real-time Apta-PCR method was used to evaluate G12 aptamer affinity acitivity.ResultsAfter 12 rounds of selection,ten unique sequences were obtained.G-quadruplex was the main motif in prediction of the secondary structure in the 8 aptamer clones selected in the random region.Sequence G12 bound to cortisol showed potential use in cortisol detection.ConclusionThe TISSD-SELEX technology is established and aptamers against cortisol are obtained.The TISSD-SELEX technology is likely to be a general aptamer-screening method for small molecule targets,without the need to be coupled in the solid-phase media or macromolecules.