Production of aptamer by SELEX technique and its application to diagnosis

SELEX技术制备核酸适体及其在诊断中的应用

基本信息

批准号：
19590571
负责人：
ARAKAWA Hidetoshi
金额：
$ 2.83万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2007
资助国家：
日本
起止时间：
2007 至 2009
项目状态：
已结题

项目摘要

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.

适体是能够以高亲和力和特异性识别多种靶分子的核酸配体，它们是通过一种称为指数富集配体系统进化（SELEX）的技术通过体外选择随机寡核苷酸池而产生的。体外选择和扩增技术可以识别与靶分子具有高亲和力的特异性适体，甚至可以区分密切相关的靶标，其结合亲和力与抗体和抗原相当。适体具有以下优点：(i) 通过化学或酶合成简单制备，(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) 易于通过微小的结合特性修饰序列的改变，（vi）实验动物的使用最小化，以及（viii）制备有毒或本身不具有免疫原性的靶标，适体适合作为需要分子识别的应用中的分析、诊断和治疗工具，因此受到越来越多的关注。其潜在用途。研究中，为了开发新的诊断方法，我们利用微芯片电泳开发了灵敏且高性能的适体结合测定法，并制备了 HMG（人绝经期促性腺激素）、β-淀粉样蛋白肽和催产素的新适体。凝血酶适配体和微芯片电泳方法，结果显示检测限为1 pmol。接下来我们通过制备HMG适配体。使用适体以类似的方式检测SELEX和HMG。另外，制备淀粉样蛋白-β肽和催产素适体。