Production of aptamer by SELEX technique and its application to diagnosis

核酸适体是一种核酸配体，具有很高的亲和力和特异性，可以识别广泛的靶分子。它们是通过一种被称为配体指数富集系统进化（SELEX）的技术，在体外选择随机寡核苷酸池产生的。体外选择和扩增技术的发展使得鉴定出具有高亲和力结合靶分子的特异性适配体，甚至可以区分密切相关的靶分子。它们的结合亲和力与抗体和抗原相当。与抗体相比，适体具有以下优点：(i)通过化学或酶合成制备简单，（ii）热稳定性，（iii）结构差异的区分，（iv）易于表面附着进行分析，(v)易于通过微小的序列改变结合特性，（vi）最小化实验动物的使用，以及（viii）制备有毒或非固有免疫原性的靶标。适配体适用于需要分子识别的应用中作为分析、诊断和治疗工具，因此其潜在用途越来越受到关注。本研究采用微芯片电泳技术，建立了灵敏、高效的适体结合检测方法，制备了新的适体，分别用于人绝经期促性腺激素（HMG）、淀粉样β肽和催产素。首先采用凝血酶适体和微芯片电泳法建立了一种检测凝血酶的方法，检测限为1 pmol。接下来，我们用SELEX制备了HMG的适体，并以类似的方法使用该适体检测HMG。此外，还制备了淀粉样β肽和催产素适体。

Aptamers are nucleic acid ligands that recognize a wide range of target molecules with high affinity and specificity. They are generated by in vitro selection of a randomized oligonucleotide pool in a technique known as systematic evolution of ligands by exponential enrichment (SELEX). The development of in vitro selection and amplification technology has allowed the identification of specific aptamers that bind target molecules with high affinity and even discriminate between closely related targets. Their binding affinities are comparable to those of antibodies and antigens. In comparison to antibodies, aptamers have the advantages of (i) simple preparation by chemical or enzymatic synthesis, (ii) thermal stability, (iii) discrimination of structural differences, (iv) ease of surface attachment for analysis, (v) ease of binding property modification through minor changes in sequence, (vi) minimization of experimental animal usage, and (viii) preparation for targets that are toxic or not inherently immunogenic. Aptamers are suitable as analytical, diagnostic, and therapeutic tools in applications requiring molecular recognition and are consequently gaining increased attention for their potential uses. In this study, to develop new diagnosis method, we developed sensitive and high performance aptamer binding assay using microchip electorophoresis, and prepared new aptamers for HMG (human menopausal gonadotropin), amyloid-β peptide and oxytocin. At first, a assay for Thronbin was developed using thrombin aptamer and microchip electrophoresis method, the result showed detection limit of 1 pmol. Next we prepared aptamer for HMG by SELEX, and HMG was detected by using the aptamer in a similar manner. In addition, amyloid-β peptide and oxytocin aptmaers were prepared.