军事医学2011，Vol.35Issue(10)：732-736,5.
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
摘要
Abstract
Objective To 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. Methods A 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. Results After 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. Conclusion The 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.关键词
SELEX/皮质醇/核酸适体/纳米技术Key words
SELEX/ cortisol/ aptamer/ nanotechnology分类
医药卫生引用本文复制引用
张岭,王新兴,战锐,弓景波,钱令嘉..靶标诱导变构解离SELEX技术筛选皮质醇特异核酸适体[J].军事医学,2011,35(10):732-736,5.基金项目
国家科技重大专项课题 (2008ZXJ090-011) （2008ZXJ090-011）
国家自然科学基金青年资助项目(81000584) （81000584）
