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用DNA修复延迟的异热CRISPR放大用于快速,敏感的基切除修复酶试验

Hyogu Han¹, Se Hee Jang², Jun Ki Ahn³

¹User Convenience Technology R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, 15588, South Korea; Department of Chemistry, Gangneung-Wonju National University, Gangneung, 25457, South Korea.

Analytica chimica acta

|June 14, 2024

概括

我们开发了UDG-REtarded CRISPR放大 (URECA),这是检测 uracil DNA glycosylase (UDG) 活动的简化试验. 乌雷卡简化了检测,为生物和临床应用提供了灵敏和可重复的方法.

科学领域:

生物化学生物化学
分子生物学分子生物学
基因组学就是基因组学.

背景情况:

乌拉DNA糖酶 (UDG) 对于保持基因组完整性至关重要.
异常的UDG活动与癌症和神经退行性疾病有关.
现有的UDG测定复杂,耗时,需要复杂的探针设计.

研究的目的:

开发一种简化,灵敏和高效的检测方法来检测UDG活动.
为了克服以前的UDG检测方法的局限性.
建立一个用于酶活性检测和药物查的多功能平台.

主要方法:

开发了UDG-RE延迟的CRISPR放大 (URECA),一个一个的异热系统.
与CRISPR/Cas12a系统相结合的同热核酸放大.
设计了一种 UDG 基质,在 uracil 切除时激活 CRISPR/Cas12a, UDG 抑制反应.

主要成果:

在50分钟内,URECA检测到UDG活性低至9.17×10−4U/mL.
在使用血和尿液样本的回收试验中实现了高精度和可重复性.
成功确定了细胞提取物中的UDG活动,并选了潜在的UDG抑制剂.

结论:

乌雷卡是一种无的,单步测定,简化了UDG检测.
消除了在探针设计中考虑PAM地点的需要,简化了测试开发.
乌雷卡原则可以扩展到为具有临床意义的各种酶创建生物传感器.

关键词:

基础切除修复的基础切除修复生物传感器是一种生物传感器.这就是CRISPR/Cas12a.酶检测试验酶检测试验异热放大器的同热放大方式 UDG UDG UDG UDG UDG UDG UDG UDG UDG UDG UDG UDG UDG UDG UDG UDG UDG UDG

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