Feng Zhi-Yuan, Yun Yang-Fang, Li Xiang, Zhang Jingjing
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China.
Chembiochem. 2023 May 16;24(10):e202300034. doi: 10.1002/cbic.202300034. Epub 2023 Apr 25.
CRISPR-LbuCas13a has emerged as a revolutionary tool for in vitro diagnosis. Similar to other Cas effectors, LbuCas13a requires Mg to maintain its nuclease activity. However, the effect of other divalent metal ions on its trans-cleavage activity remains less explored. Herein, we addressed this issue by combining experimental and molecular dynamics simulation analysis. In vitro studies showed that both Mn and Ca could replace Mg as cofactors of LbuCas13a. In contrast, Ni , Zn , Cu , or Fe inhibits the cis- and trans-cleavage activity, while Pb does not affect it. Importantly, molecular dynamics simulations confirmed that calcium, magnesium, and manganese hydrated ions have a strong affinity to nucleotide bases, thus stabilizing the conformation of crRNA repeat region and enhancing the trans-cleavage activity. Finally, we showed that combination of Mg and Mn can further enhance the trans-cleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis.
CRISPR-LbuCas13a已成为一种用于体外诊断的革命性工具。与其他Cas效应物类似，LbuCas13a需要镁来维持其核酸酶活性。然而，其他二价金属离子对其反式切割活性的影响仍有待深入研究。在此，我们通过结合实验和分子动力学模拟分析来解决这个问题。体外研究表明，锰和钙都可以替代镁作为LbuCas13a的辅因子。相比之下，镍、锌、铜或铁会抑制顺式和反式切割活性，而铅则不影响其活性。重要的是，分子动力学模拟证实，钙、镁和锰水合离子对核苷酸碱基具有很强的亲和力，从而稳定了crRNA重复区域的构象并增强了反式切割活性。最后，我们表明镁和锰的组合可以进一步增强反式切割活性，以实现扩增RNA检测，揭示了其在体外诊断中的潜在优势。
