采用β-1,3-葡萄糖识别蛋白标记蛋白和珠选用aptamer的选择
Kazuyuki Kumagai1, Hiroki Okubo1, Ryo Amano1
1Department of Life Science, Faculty of Advanced Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
Journal of biochemistry
|July 27, 2023
概括
这项研究引入了一种新的SELEX方法,使用GRP标签和curdlan珠用于特定的RNA吸收酶选择. 这种方法有效地减少了非特异性结合,为像AML1 Runt域这样的目标蛋白产生高质量的体.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
背景情况:
- 通过指数式丰富对联体的系统进化 (SELEX) 是一种用于体发现的方法.
- 传统的SELEX方法可以产生与蛋白标签和固定矩阵结合的aptamers,从而降低特异性.
- 蛋白质标签和矩阵可以干扰aptamer-target的相互作用.
研究的目的:
- 开发和评估一种新的SELEX策略,使用β-1,3-葡萄糖识别蛋白 (GRP) 标签和curdlan珠用于特定的aptamer选择.
- 使用这种新方法,评估针对急性骨髓性白血病1 (AML1) 产生的aptamers的特异性.
- 为了比较GRP标签与传统His标签在SELEX中的有效性.
主要方法:
- 使用 GRP 标记的 AML1 Runt 域固定在 curdlan 珠子上执行 SELEX.
- 分析了使用高通量测序的aptamer丰富.
- 利用表面等离子体共振来确认阿普坦特异性和结合亲和力.
主要成果:
- 使用GRP标签的SELEX成功丰富了与His标签相似的体.
- 生成的阿普坦没有与GRP标签结合.
- 将GRP标签与Runt域的结合削弱了aptamer-target的相互作用,这表明它具有竞争性的作用.
结论:
- 对于SELEX,GRP-tag和curdlan珠子系统是有效的,产生特定的RNA体.
- 这种亲和系统最大限度地减少了非特异性结合,改善了阿普坦质量.
- GRP标签可以作为竞争对手来增强高亲和度体的选择.
相关概念视频
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02:57Southern Blot
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