<i>Pyrococcus furiosus</i> Argonaute-mediated nucleic acid detection

He, Ruyi; Wang, Longyu; Wang, Fei; Li, Wenqiang; Liu, Yang; Li, Aitao; Wang, Yuan; Mao, Weihua; Zhai, Chao

doi:10.1039/c9cc07339f

Cited by 82 publications

(76 citation statements)

References 24 publications

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“…In particular, degradation of one of the target variants can be used for enrichment of rare nucleic acid variants, both DNA and RNA, for their further detection. Thermophilic TtAgo and PfAgo were previously applied for precise recognition and cleavage of complementary targets and their closely related variants depending on the presence of mismatches ( 34 , 35 ). Similarly, thermophilic MpAgo, programmed with RNA guides, was used for selective binding of rare RNA variants with single-nucleotide changes without their cleavage ( 31 ).…”

Section: Discussionmentioning

confidence: 99%

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

Kropocheva

Kuzmenko

Aravin

et al. 2021

Nucleic Acids Research

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Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique pAgo nuclease, KmAgo, from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo binds 16–20 nt long 5′-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acids. Target RNA cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.

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Section: Discussionmentioning

confidence: 99%

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

Kropocheva

Kuzmenko

Aravin

et al. 2021

Nucleic Acids Research

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“…Expression and purification of the recombinant PfAgo was performed as previously described ( He et al, 2019 ). RT-PCR mixture in a final volume of 25 μl was prepared as previously described.…”

Section: Methodsmentioning

confidence: 99%

“…As a nucleic-acid-guided endonuclease, PfAgo prefers to cleave DNA substrate under the guidance of short 5′-phosphorylated single-strand DNA without the presence of protospacer-adjacent motif (PAM). In 2019, we developed a P f A go ( Pyrococcus furiosus Argonaute) mediated n ucleic acid d etection method (PAND) ( He et al, 2019 ). In the present study, we simplified PAND and applied it on the detection of SARS-CoV-2 and its variants ( Fig.…”

Section: Introductionmentioning

confidence: 99%

PfAgo-based detection of SARS-CoV-2

Wang

Yang

et al. 2021

Biosensors and Bioelectronics

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“…By mining the expanding repertoire of microbial genome sequences, many pAgo genes have been reported, such as Thermus thermophilus (TtAgo) [ 4 , 12 , 21 ], archaeon Pyrococcus furiosus (PfAgo) [ 1 , 22 ], archaeal organism Methanocaldococcus jannaschii (MjAgo) [ 5 , 23 , 24 ], CRISPR-associated Marinitoga piezophila (MpAgo) [ 11 , 25 , 26 ], Clostridium butyricum (CbAgo) [ 2 , 13 , 14 , 27 ], Rhodobacter sphaeroides (RsAgo) [ 28 , 29 ], and Natronobacterium gregoryi (NgAgo) [ 15 , 16 ]. These pAgos have been partially characterized and demonstrated to function as nucleases with different properties in terms of guide-binding and target-processing.…”

Section: Introductionmentioning

confidence: 99%

Expression and Functional Analysis of the Argonaute Protein of Thermus thermophilus (TtAgo) in E. coli BL21(DE3)

Xing

Cheng

et al. 2021

Biomolecules

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The prokaryotic Argonaute proteins (pAgos) have been reported to cleave or interfere with DNA targets in a guide-dependent or independent manner. It is often difficult to characterize pAgos in vivo due to the extreme environments favored by their hosts. In the present study, we expressed functional Thermus thermophilus pAgo (TtAgo) in E. coli BL21 (DE3) cells at 37 °C. Initial attempts to express TtAgo in BL21(DE3) cells at 37 °C failed. This was not because of TtAgo mediated general toxicity to the host cells, but instead because of TtAgo-induced loss of its expression plasmid. We employed this discovery to establish a screening system for isolating loss-of-function mutants of TtAgo. The E. colifabI gene was used to help select for full-length TtAgo loss of function mutants, as overexpression of fabI renders the cell to be resistant to the triclosan. We isolated and characterized eight mutations in TtAgo that abrogated function. The ability of TtAgo to induce loss of its expression vector in vivo at 37 °C is an unreported function that is mechanistically different from its reported in vitro activity. These results shed light on the mechanisms by which TtAgo functions as a defense against foreign DNA invasion.

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Pyrococcus furiosus Argonaute-mediated nucleic acid detection

Abstract: PfAgo-mediated Nucleic acid Detection (PAND) distinguishes single-nucleotide mutants and accomplishes multiplexed detection by a second round of cleavage.

Cited by 82 publications

References 24 publications

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

PfAgo-based detection of SARS-CoV-2

Expression and Functional Analysis of the Argonaute Protein of Thermus thermophilus (TtAgo) in E. coli BL21(DE3)

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