An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate Spectrum
Abstract PfAgo, a thermophilic Argonaute nuclease from Pyrococcus furiosus, is widely used in various fields due to its high DNA‐guided DNA cleavage activity. However, its high‐temperature‐dependent cleavage activity largely restricts its applications in moderate‐temperature scenarios. In this study...
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Wiley
2025-08-01
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| Series: | Advanced Science |
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| Online Access: | https://doi.org/10.1002/advs.202416631 |
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| author | Longyu Wang Xiaochen Xie Fuyong Huang Qiang Wei Tianxin Cai Na Yu Shi Chen Fei Wang Wanping Chen Chin‐yu Chen Chunhua Li Lixin Ma |
| author_facet | Longyu Wang Xiaochen Xie Fuyong Huang Qiang Wei Tianxin Cai Na Yu Shi Chen Fei Wang Wanping Chen Chin‐yu Chen Chunhua Li Lixin Ma |
| author_sort | Longyu Wang |
| collection | DOAJ |
| description | Abstract PfAgo, a thermophilic Argonaute nuclease from Pyrococcus furiosus, is widely used in various fields due to its high DNA‐guided DNA cleavage activity. However, its high‐temperature‐dependent cleavage activity largely restricts its applications in moderate‐temperature scenarios. In this study, PfAgo is engineered for cold adaptation based on its ternary complex structure and the attributes of cold‐adapted enzymes, yielding a series of variants with better performance at moderate temperatures. Among those, mPfAgo (K617G, L618G) exhibits significantly promoted cleavage activity at 37 °C and a wider catalytic temperature range of 30–95 °C. Its high‐temperature cleavage activity is also greatly improved, enabling its application in DNA detection with attomolar sensitivity in the presence of Mg2+. Additionally, mPfAgo shows versatile cleavage activities, including DNA cleavage guided by 5′OH‐gDNA, 5′P‐gDNA, or 5′COOH‐gDNA, as well as RNA cleavage with 5′OH‐gDNA, 5′P‐gDNA, 5′P‐gRNA, or 5′COOH‐gDNA as guides. Further analysis through far‐UV CD spectra and DSF indicates that mPfAgo has a more flexible structure than wild‐type PfAgo. Furthermore, this established strategy is applied to engineer TtdAgo, likewise obtaining its variants with enhanced moderate‐temperature activity and expanded substrate spectrum. In summary, this work provides a novel method for the rational design of thermophilic Agos, thereby greatly expanding their application scopes. |
| format | Article |
| id | doaj-art-e14faba342fc442c861e67963e11e30a |
| institution | DOAJ |
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| language | English |
| publishDate | 2025-08-01 |
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| series | Advanced Science |
| spelling | doaj-art-e14faba342fc442c861e67963e11e30a2025-08-20T02:57:53ZengWileyAdvanced Science2198-38442025-08-011229n/an/a10.1002/advs.202416631An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate SpectrumLongyu Wang0Xiaochen Xie1Fuyong Huang2Qiang Wei3Tianxin Cai4Na Yu5Shi Chen6Fei Wang7Wanping Chen8Chin‐yu Chen9Chunhua Li10Lixin Ma11State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences Hubei University Wuhan Hubei 430062 ChinaAbstract PfAgo, a thermophilic Argonaute nuclease from Pyrococcus furiosus, is widely used in various fields due to its high DNA‐guided DNA cleavage activity. However, its high‐temperature‐dependent cleavage activity largely restricts its applications in moderate‐temperature scenarios. In this study, PfAgo is engineered for cold adaptation based on its ternary complex structure and the attributes of cold‐adapted enzymes, yielding a series of variants with better performance at moderate temperatures. Among those, mPfAgo (K617G, L618G) exhibits significantly promoted cleavage activity at 37 °C and a wider catalytic temperature range of 30–95 °C. Its high‐temperature cleavage activity is also greatly improved, enabling its application in DNA detection with attomolar sensitivity in the presence of Mg2+. Additionally, mPfAgo shows versatile cleavage activities, including DNA cleavage guided by 5′OH‐gDNA, 5′P‐gDNA, or 5′COOH‐gDNA, as well as RNA cleavage with 5′OH‐gDNA, 5′P‐gDNA, 5′P‐gRNA, or 5′COOH‐gDNA as guides. Further analysis through far‐UV CD spectra and DSF indicates that mPfAgo has a more flexible structure than wild‐type PfAgo. Furthermore, this established strategy is applied to engineer TtdAgo, likewise obtaining its variants with enhanced moderate‐temperature activity and expanded substrate spectrum. In summary, this work provides a novel method for the rational design of thermophilic Agos, thereby greatly expanding their application scopes.https://doi.org/10.1002/advs.202416631cold adaptationnucleasesPfAgorational engineeringthermophilic archaea |
| spellingShingle | Longyu Wang Xiaochen Xie Fuyong Huang Qiang Wei Tianxin Cai Na Yu Shi Chen Fei Wang Wanping Chen Chin‐yu Chen Chunhua Li Lixin Ma An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate Spectrum Advanced Science cold adaptation nucleases PfAgo rational engineering thermophilic archaea |
| title | An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate Spectrum |
| title_full | An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate Spectrum |
| title_fullStr | An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate Spectrum |
| title_full_unstemmed | An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate Spectrum |
| title_short | An Engineered PfAgo with Wide Catalytic Temperature Range and Substrate Spectrum |
| title_sort | engineered pfago with wide catalytic temperature range and substrate spectrum |
| topic | cold adaptation nucleases PfAgo rational engineering thermophilic archaea |
| url | https://doi.org/10.1002/advs.202416631 |
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