AlphaFold 3 sheds insights into chemical enhancer-induced structural changes in Cas12a RNPs

作     者：Lulu Pan Aifeng Wang Yongcheng Ma Rui Sang Fei Deng Xia Lu Wenjie Chen 

作者机构：Central China Fuwai Hospital of Zhengzhou University Henan Provincial People’s Hospital Zhengzhou 450003 China Graduate School of Biomedical Engineering Faculty of Engineering ARC Centre of Excellence in Nanoscale Biophotonics UNSW Sydney Sydney 2052 Australia The Key Lab of Animal Disease and Public Health Henan University of Science and Technology 263 Kaiyuan Avenue Luoyang 471023 China Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control Luoyang 471023 China Luoyang Worpson Biological Engineering Co Luolong Industrial Zone 18 Zimei Rd LtdLuoyang 471000 No China Guangdong Province & NMPA & State Key Laboratory School of Pharmaceutical Sciences Department of Emergency The Second Affiliated Hospital Guangzhou Medical University Guangzhou 511436 China 

出 版 物：《Health Nanotechnology》 

年 卷 期：2025年第1卷第1期

页      面：1-11页

摘      要：Background The CRISPR/Cas12a system has revolutionized nucleic acid detection through trigger-activated nonspecific trans-cleavage activity. Enhancing this activity is vital for improving the sensitivity of CRISPR/Cas biosensing systems. Although various chemical enhancers have been shown to affect Cas12a ribonucleoprotein (RNP), the underlying mechanisms remain poorly understood. Investigating how these enhancers alter the structure of Cas12a RNPs is essential for elucidating the enhancement mechanisms involved. Results This study focuses on elucidating the structural changes in Cas12a RNPs induced by various chemical enhancers via AlphaFold 3, an emerging and powerful tool for analyzing structural changes in protein‒nucleic acid complexes. We validated the ability of AlphaFold 3 to simulate structural changes in Cas12a RNPs activated by triggers and subsequently analyzed the effects of specific enhancers, such as reducing agents (e.g., Dithiothreitol, namely DTT), divalent cations (e.g., Mg2+, Mn2+), and bovine serum albumin (BSA). Our findings revealed that DTT, simulated with a cysteine-to-serine Cas12a mutant, caused significant structural changes in Cas12a RNP, as evidenced by notable shifts in the distance between key residues (Val377 to Gln1136) and a high root mean square deviation (RMSD)(RMSD 2). Conversely, divalent cations and BSA did not cause substantial structural changes, resulting in only minor shifts in residue distance and a low RMSD (RMSD Conclusions These results demonstrate that DTT enhances Cas12a activity by inducing significant structural rearrangements, whereas divalent cations and BSA-induced enhancements do not involve substantial structural modifications. h3