毛细管电泳筛选盐酸克伦特罗核酸适配体的方法研究

Screening of Clenbuterol Hydrochloride Aptamers Based on Capillary Electrophoresis

毛细管电泳(CE)被认为是核酸适配体筛选的高效方法,其无需介质固定,在溶液相中完成筛选,分离高效,样品用量少,筛选成本低。但由于小分子靶与核酸的结合位点少,且小分子的核酸复合物与核酸库的电泳迁移率差异小,通常认为CE-SELEX不适用于筛选小分子靶标的适配体。本研究建立了基于CESELEX的盐酸克伦特罗靶标的的适配体筛选方法。将80 nt的ssDNA库与盐酸克伦特罗特混合孵育,通过CZE-UV分离混合物。收集ssDNA库峰前的馏分,通过PCR扩增和ssDNA制备,完成第一轮筛选。将次级核酸库经过3轮筛选,获得10条ssDNA序列。选择3条亲和力较强的序列Apt 4、Apt 7、Apt 12,通过CE-LIF测定其与盐酸克伦特罗的K_d分别为9.315×10^(-7)mol/L、1.040×10^(-6)mol/L和1.143×10^(-5)mol/L。软件分析表明,上述3条序列均可形成茎环二级结构,其中Apt 4茎环结构的自由能最低,结构最稳定。以沙丁胺醇为对照,验证了3条序列,具有较高的特异性。

Capillary electrophoresis based systematic evolution of ligands via exponential enrichment(CE-SELEX)was reported as a homogeneous efficient method for high-affinity selection of aptamer,with several merits involving screening in free solution without nonspecific binding,capable of high-efficient separation,low-sample consumption,and saving money.There are few studies regarding the aptamer selection against small molecule using CE-SELEX,resulting from the aspects of less binding sites and the negligible variety of its complex with nucleic acid in the electrophoretic mobility.In this study,the aptamer selection was performed towards a small molecule target of clenbuterol hydrochloride(Cle)by CE-SELEX.In brief,incubated Cle and an 80 nt ssDNA library were firstly incubated,and CZE-UV approach was used to separate complex and random ssDNA.The complex was then collected into a vial followed by PCR amplification.Through three round selections,the third pool was selected to clone and ten sequences were finally obtained.The dissociation constants(K d)of three potential candidates(Apt 4,Apt 7 and Apt 12)were determined by CE-LIF,and showed high affinities of 9.315×10^-7 mol/L,1.040×10^-6 mol/L and 1.143×10^-5 mol/L,respectively.The m-Fold software analysis showed that the above three sequences could form stem-loop structure,and Apt 4 gave the lowest free energy and the most stable structure.Using salbutamol as a control,three selected aptamers were verified with high specificity.