Production of aptamer by SELEX technique and its application to diagnosis

适配子是识别多种靶分子的核酸配体，具有很高的亲和力和特异性。它们是通过指数富集法(SELEX)在体外选择随机寡核苷酸池产生的，这种技术称为配体系统进化(SELEX)。体外选择和扩增技术的发展使得能够以高亲和力结合靶分子甚至区分密切相关的靶分子的特定适配子的鉴定成为可能。它们的结合亲和力与抗体和抗原的亲和力相当。与抗体相比，适配子具有以下优点：(I)通过化学或酶合成制备简单，(Ii)热稳定性，(Iii)区分结构差异，(Iv)易于表面附着以进行分析，(V)通过微小的序列改变易于结合性质改变，(Vi)最大限度地减少实验动物的使用，以及(Viii)制备有毒或非固有免疫原性的靶标。适体在需要分子识别的应用中作为分析、诊断和治疗工具是合适的，因此它们的潜在用途得到了越来越多的关注。为了开发新的诊断方法，我们利用微芯片电泳法建立了灵敏、高效的核酸适体结合分析方法，并制备了人绝经期促性腺激素、淀粉样蛋白-β多肽和催产素的新适配子。首先，利用凝血酶适配子和微芯片电泳法建立了凝血酶的检测方法，检测下限为1pmol。接下来，我们用SELEX法制备了HMG的适体，并用同样的方法检测了HMG。此外，还制备了淀粉样蛋白-β多肽和催产素适体。

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.