用于编码和解码锁定核酸序列的高进程性和精度的DNA聚合酶变体
Hidekazu Hoshino1,2, Yuuya Kasahara1,2, Masayasu Kuwahara3
1National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Journal of the American Chemical Society
|December 11, 2020
概括
研究人员设计了KOD DNA聚合酶变体,以有效合成和转录锁定核酸 (LNA),这是一个有前途的治疗分子. 这些工程聚合酶使得创建新型LNA体和催化剂成为可能,进步了核酸疗法.
科学领域:
- 生物化学
- 分子生物学
- 生物技术
背景情况:
- 异生物核酸 (XNA) 是DNA和RNA的化学修饰类型,具有治疗潜力.
- 锁定核酸 (LNA) 是一个有前途的XNA,但由于缺乏高效的合成和反转录工具,其治疗发展受到阻碍.
- 现有的LNA胺和催化剂开发方法需要专门的聚合酶来进行DNA-LNA合成和LNA-DNA逆转录.
研究的目的:
- 开发能够有效合成LNA (编码) 和逆转录 (解码) 的工程DNA聚合酶.
- 提高KODDNA聚合酶的性能,以制造基于LNA的治疗方法.
- 为了能够合成长的LNA序列,并适应2'-O-甲基 (2'-OMe) 等常见的治疗修饰.
主要方法:
- 对KODDNA聚合酶进行结构分析以指导蛋白质工程.
- 局部定向的突变产生LNA解码和编码KOD DNA聚合酶的变体.
- 工程聚合酶的效率,准确性和基质接受性的实验验证,包括基基长度LNA和LNA合物的SELEX合成.
主要成果:
- 两种KOD DNA聚合酶变体,KOD DGLNK (DNA → LNA) 和KOD DLK (LNA → DNA) 已成功设计.
- 这两种变体在LNA合成和逆转录方面显著提高了效率和准确性.
- 使用KOD DGLNK合成了千基基长的LNA,这些变体接受了2'-O-甲基修饰,这对于治疗应用至关重要.
结论:
- 工程 KOD DNA 聚合酶变体为 LNA 合成和逆转录提供了强大的工具.
- 这些变体克服了先前的局限性,促进了新型LNA体和催化剂的开发.
- 开发的技术通过使自然DNA和RNA不同分子的创建,推动了XNA治疗领域的发展.
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