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使用分子动力学模拟研究基对不匹配对Cas13d裂变效率的影响

Ye Liu^1,2, Yan Li^1,2, Guohui Li^1,2

¹Interdisciplinary Research Center for Biology and Chemistry, Liaoning Normal University, Dalian 116029, China.

The journal of physical chemistry. B

|October 18, 2025

概括

目标RNA中的不匹配会破坏未培养的Ruminococcus sp. Cas13d (UrCas13d) 活动通过改变域结构和埋葬催化中心. 这阻止了目标RNA的结合,降低了裂变效率,并为更特定的Cas13d变体的设计提供了信息.

科学领域:

分子生物学分子生物学
生物化学生物化学
基因工程是一种基因工程.

背景情况:

CRISPR-Cas13d是一种多功能RNA编辑工具,具有基因工程的潜力.
Cas13d对目标RNA不匹配的敏感性限制了其在核酸测试和基因治疗中的应用.
了解不匹配减少Cas13d活动的机制对于提高其特异性至关重要.

研究的目的:

为了研究背后的分子机制不匹配诱导非培养的Ruminococcus sp.的无活化. 在Cas13d (UrCas13d).
为了阐明目标RNA不匹配如何影响UrCas13d.d的结构动态.
为设计更具体的Cas13d变体提供理论见解.

主要方法:

用分子动力学 (MD) 模拟来研究UrCas13d-RNA相互作用.
对双重解卷,域相互作用和构造性重排的分析.
调查HEPN域内的残留物流动及其对催化中心的影响.

主要成果:

目标RNA的不匹配会导致RNA复合体的解和扭曲,增加与螺旋域的相互作用.
这些相互作用阻碍了激活所需的螺旋-1和螺旋-2域的形状重排.
不匹配诱导的运动埋葬了催化中心,阻止R-X4-H动机与目标RNA接触,从而降低了分裂效率.

结论:

阻止域过渡到活性构造和防止催化残留-点RNA接触是导致不匹配的UrCas13d失活的关键.
这些发现为Cas13d与不匹配的目标的减少活性提供了分子基础.
这项研究为开发具有增强特异性和减少目标外影响的工程 Cas13d 变体提供了理论支持.

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