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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.

适体是以高亲和力和特异性识别广泛范围的靶分子的核酸配体。它们通过在称为指数富集配体系统进化（SELEX）的技术中体外选择随机寡核苷酸池来产生。体外选择和扩增技术的发展已经允许鉴定以高亲和力结合靶分子并且甚至区分密切相关的靶的特异性适体。它们的结合亲和力与抗体和抗原的结合亲和力相当。与抗体相比，适体具有以下优点：（i）通过化学或酶促合成的简单制备，（ii）热稳定性，（iii）结构差异的区分，（iv）易于表面附着以用于分析，（V）易于通过序列的微小改变来修饰结合性质，（vi）最小化实验动物使用，和（viii）制备毒性或非固有免疫原性的靶。适体在需要分子识别的应用中适合作为分析、诊断和治疗工具，因此其潜在用途越来越受到关注。本研究利用微芯片电泳技术，建立了一种灵敏、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（human menopausal gonadotropin，HMG）、β淀粉样蛋白（amyloid-β peptide，AP）和催产素（oxytocin，OT）的核酸适体。首先，利用凝血酶适配体和微芯片电泳技术建立了凝血酶的检测方法，检测限为1 pmol。接下来，我们通过SELEX制备HMG的适体，并且以类似的方式通过使用适体检测HMG。此外，还制备了β淀粉样肽和催产素适配体。