SELEX技术筛选甲基苯丙胺适配子

In Vitro Selection of DNA Aptamers to Methamphetamine by SELEX

目的利用SELEX筛选甲基苯丙胺适配子,并初步鉴定获得甲基苯丙胺的适配子。方法体外合成全长为76 bp的随机ssDNA文库,以甲基苯丙胺完全抗原偶联到溴化氰活化琼脂糖上,作为固相靶分子对随机ssDNA文库进行SELEX筛选。利用SPR测定适配子和甲基苯丙胺的结合率和特异性,通过MFOLD分析软件对适配子进行二级结构预测和结合位点分析。结果经过10轮筛选后,甲基苯丙胺适配子的结合率由88.3 RU上升到113.7 RU,表明特异性适配子得到逐步富集。获得的适配子进行测序,得10个序列,二级结构均为茎-环结构,可能是适配子和甲基苯丙胺结合的结构基础。结论本研究使用SELEX技术筛选小分子毒品甲基苯丙胺适配子的方法,获得与甲基苯丙胺特异性结合的适配子序列,揭示了适配子与甲基苯丙胺结合的基本结构,为制备特异性甲基苯丙胺检测试剂盒奠定了基础。

Objective To screen and identify high-affinity aptamers to methamphetamine by SELEX （System Evolution of Lig- ands by Exponential Enrichment） method. Methods Methamphetamine-BSA was conjugated to cyanogen bromide-activated- sepharose as the solid phase target, and a synthetic random DNA library with the total length of 76 bp was subjected to selec- tion by SELEX technology. Positive aptamers from the selection were analyzed for their specific association with metham- phetamine-BSA by SPR （Surface Plasmon Resonance） sensor. The secondary structures of aptamers and their corresponding binding sites were predicted and analyzed by MFOLD software. Results After 10 rounds of SELEX screening, the binding affinity between the aptamers and methamphetamine increased from 88.3RU to 113.7RU, indicating that specific aptamers were enriched progressively. Ten aptamers have an absolute identical sequence after cloning and sequencing. It was revealed that their secondary structures were stem-loop, which might be the possible binding sites between the aptamers and the target. Conclusion The study obtained the sequence of specific aptamers to methamphetamine, and revealed the basic structure of the binding sites, which paves the way for the future application of these aptamers in a fast and cost-efficient detection of methamphetamine.