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.

APATMER是核酸配体，它们识别具有高亲和力和特异性的广泛靶标分子。它们是通过在一种被指数富集（SELEX）（SELEX）的技术进化的技术中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允许鉴定具有高亲和力的靶标分子甚至在紧密相关的靶标之间结合靶标分子的特定适体。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，适体具有（i）通过化学或酶合成简单制备的优点，（ii）热稳定性，（iii）区分结构差异，（iii）（iv）表面易于分析的易于分析，（v）通过序列的较小变化（vi）的实验用途（vi）的限制性变化（vi），（v）的实验用途（vi）的实验用途，以及（vi）的实验。或不是固有的免疫原性。在需要分子识别的应用中，APAMER适用于分析，诊断和治疗工具，因此对它们的潜在用途产生了越来越多的关注。在这项研究中，为了开发新的诊断方法，我们使用微芯片电泳开发了敏感和高性能的猿猴结合测定法，并为HMG（人类绝经性促性腺激素），淀粉样蛋白β肽和氧气制备了新的apatamers。首先，使用凝血酶凋亡和微芯片电泳方法开发了链龙宾的测定法，结果显示了1 pmol的检测极限。接下来，我们由SELEX准备了HMG的Apattern，并以类似的方式使用Apattern检测到HMG。另外，制备了淀粉样蛋白β肽和氧气适体。