Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment.

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.

Development of an optimized RPA-PfAgo detection system for MTHFR C677T polymorphism genotyping.

This study introduces an efficient RPA-PfAgo detection system for the MTHFR C677T polymorphism, proposing a potential strategy to simplify the genotyping process. By optimizing recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) nucleases, we achieved DNA amplification at a constant temperature. The assay was fine-tuned through meticulous primer and guide DNA selection, with optimal conditions established at 2.0 µL of MgAc, a reaction temperature of 42 °C, and a 10-minute reaction time for RPA. Further optimization of the PfAgo cleavage assay revealed the ideal concentrations of MnCl2, guide DNA, molecular beacon probes, the PfAgo enzyme, and the RPA product to maximize sensitivity and specificity. Clinical validation of 20 samples showed 100% concordance with Sanger sequencing, confirming the method's precision. The RPA-PfAgo system is a promising tool for on-site genotyping, with broad applications in personalized medicine and disease prevention.

A Fast and Sensitive One-Tube SARS-CoV-2 Detection Platform Based on RTX-PCR and Pyrococcus furiosus Argonaute.

Since SARS-CoV-2 is a highly transmissible virus, alternative reliable, fast, and cost-effective methods are still needed to prevent virus spread that can be applied in the laboratory and for point-of-care testing. Reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) is currently the gold criteria for detecting RNA viruses, which requires reverse transcriptase to reverse transcribe viral RNA into cDNA, and fluorescence quantitative PCR detection was subsequently performed. The frequently used reverse transcriptase is thermolabile; the detection process is composed of two steps: the reverse transcription reaction at a relatively low temperature, and the qPCR performed at a relatively high temperature, moreover, the RNA to be detected needs to pretreated if they had advanced structure. Here, we develop a fast and sensitive one-tube SARS-CoV-2 detection platform based on Ultra-fast RTX-PCR and Pyrococcus furiosus Argonaute-mediated Nucleic acid Detection (PAND) technology (URPAND). URPAND was achieved ultra-fast RTX-PCR process based on a thermostable RTX (exo-) with both reverse transcriptase and DNA polymerase activity. The URPAND can be completed RT-PCR and PAND to detect nucleic acid in one tube within 30 min. This method can specifically detect SARS-CoV-2 with a low detection limit of 100 copies/mL. The diagnostic results of clinical samples with one-tube URPAND displayed 100% consistence with RT-qPCR test. Moreover, URPAND was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The URPAND platform is rapid, accurate, tube closed, one-tube, easy-to-operate and free of large instruments, which provides a new strategy to the detection of SARS-CoV-2 and other RNA viruses.

Hotspot Wizard-informed engineering of a hyperthermophilic ß-glucosidase for enhanced enzyme activity at low temperatures.

Hyperthermophilic enzymes serve as an important source of industrial enzymes due to their high thermostability. Unfortunately, most hyperthermophilic enzymes suffer from reduced activity at low temperatures (e.g., ambient temperature), limiting their applicability. In addition, evolving hyperthermophilic enzymes to increase low temperature activity without compromising other desired properties is generally difficult. In the current study, a variant of ß-glucosidase from Pyrococcus furiosus (PfBGL) was engineered to enhance enzyme activity at low temperatures through the construction of a saturation mutagenesis library guided by the HotSpot Wizard analysis, followed by its screening for activity and thermostability. From this library construction and screening, one PfBGL mutant, PfBGL-A4 containing Q214S/A264S/F344I mutations, showed an over twofold increase in ß-glucosidase activity at 25 and 50°C compared to the wild type, without compromising high-temperature activity, thermostability and substrate specificity. Our experimental and computational characterizations suggest that the findings with PfBGL-A4 may be due to the elevation of local conformational flexibility around the active site, while slightly compacting the global protein structure. This study showcases the potential of HotSpot Wizard-informed engineering of hyperthermophilic enzymes and underscores the interplays among temperature, enzyme activity, and conformational flexibility in these enzymes.

PfAgo-Based Zika Virus Detection.

As a mosquito-borne flavivirus, Zika virus (ZIKV) has been identified as a global health threat. The virus has been linked to severe congenital disabilities, including microcephaly and other congenital malformations, resulting in fatal intrauterine death. Therefore, developing sensitive and specific methods for the early detection and accurate diagnosis of the ZIKV is essential for controlling its spread and mitigating its impact on public health. Herein, we set up a novel nucleic acid detection system based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection, targeting the non-structural protein 5 (NS5) region of the ZIKV genome (abbreviated ZIKV-PAND). Without preamplification with the polymerase chain reaction (PCR), the minimum detection concentration (MDC) of ZIKV-PAND was about 10 nM. When introducing an amplification step, the MDC can be dramatically decreased to the aM level (8.3 aM), which is comparable to qRT-PCR assay (1.6 aM). In addition, the diagnostic findings from the analysis of simulated clinical samples or Zika virus samples using ZIKV-PAND show a complete agreement of 100% with qRT-PCR assays. This correlation can aid in the implementation of molecular testing for clinical diagnoses and the investigation of ZIKV infection on an epidemiological scale.

Pyrococcus furiosus Argonaute Based Detection Assays for Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a major cause of diarrhea and diarrhea-related deaths among piglets and results in massive losses to the overall porcine industry. The clinical manifestations of porcine diarrhea brought on by the porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and PDCoV are oddly similar to each other. Hence, the identification of different pathogens through molecular diagnosis and serological techniques is crucial. Three novel detection methods for identifying PDCoV have been developed utilizing recombinase-aided amplification (RAA) or reverse transcription recombinase-aided amplification (RT-RAA) in conjunction with Pyrococcus furiosus Argonaute (PfAgo): RAA-PfAgo, one-pot RT-RAA-PfAgo, and one-pot RT-RAA-PfAgo-LFD. The indicated approaches have a detection limit of around 60 copies/µL of PDCoV and do not cross-react with other viruses including PEDV, TGEV, RVA, PRV, PCV2, or PCV3. The applicability of one-pot RT-RAA-PfAgo and one-pot RT-RAA-PfAgo-LFD were examined using clinical samples and showed a positive rate comparable to the qPCR method. These techniques offer cutting-edge technical assistance for identifying, stopping, and managing PDCoV.

Recombinase polymerase amplification combined with Pyrococcus furiosus Argonaute for fast Salmonella spp. testing in food safety.

Foodborne illness caused by Salmonella spp. is one of the most prevalent public health problems globally, which have brought immeasurable economic burden and social impact to countries around the world. Neither current nucleic acid amplification detection method nor standard culture method (2-3 days) are suitable for field detection in areas with a heavy burden of Salmonella spp. Here, we developed a highly sensitive and accurate assay for Salmonella spp. detection in less than 40 min. Specifically, the invA gene of Salmonella spp. was amplified by recombinase polymerase amplification (RPA), followed by Pyrococcus furiosus Argonaute (PfAgo)-based target sequence cleavage, which could be observed by a fluorescence reader or the naked eye. The assay offered the lowest detectable concentration of 1.05 × 101 colony forming units/mL (CFU/mL). This assay had strong specificity and high sensitivity for the detection of Salmonella spp. in field samples, which indicated the feasibility of this assay.

Fast and Ultrasensitive Detection of Monkeypox by a Pyrococcus furiosus Argonaute System Coupled with a Short Amplification.

Monkeypox virus (MPXV), the pathogen responsible for the infectious disease monkeypox, causes lesions on the skin, lymphadenopathy, and fever. It has posed a global public health threat since May 2022. Highly sensitive and specific detection of MPXV is crucial for preventing the spread of the disease. Pyrococcus furiosus Argonaute (PfAgo) is an artificial DNA-guided restriction cleavage enzyme programmable with 5'-phosphorylated ssDNA sequences, which can be developed to specifically detect nucleic acids of pathogens. Here, a PfAgo-based system was established for the detection of MPXV-specific DNA targeting the F3L gene. A short amplicon of 79 bp could be obtained through a fast PCR procedure, which was completed within 45 min. Two 5'-phosphorylation guide DNAs were designed to guide PfAgo to cleave the amplicon to obtain an 18 bp 5'-phosphorylation sequence specific to MPXV, not to other orthopoxviruses (cowpox, variola, and vaccinia viruses). The 18 bp sequence guided PfAgo to cleave a designed probe specific to MPXV to emit fluorescence. With optimized conditions for the PfAgo-MPXV system, it could be completed in 60 min for the detection of the extracted MPXV DNA with the limit of detection (LOD) of 1.1 copies/reaction and did not depend on expensive instruments. Successful application of the PfAgo-MPXV system in sensitively detecting MPXV in simulated throat swabs, skin swabs, sera, and wastewater demonstrated the system's good performance. The PfAgo platform, with high sensitivity and specificity established here, has the potential to prevent the spread of MPXV.

Effects of heterologous expression and N-glycosylation on the hyperthermostable endoglucanase of Pyrococcus furiosus.

Hyperthermostable endoglucanases of glycoside hydrolase family 12 from the archaeon Pyrococcus furiosus (EGPf) catalyze the hydrolysis of ß-1,4-glucosidic linkages in cellulose and ß-glucan structures that contain ß-1,3- and ß-1,4-mixed linkages. In this study, EGPf was heterologously expressed with Aspergillus niger and the recombinant enzyme was characterized. The successful expression of EGPf resulted as N-glycosylated protein in its secretion into the culture medium. The glycosylation of the recombinant EGPf positively impacted the kinetic characterization of EGPf, thereby enhancing its catalytic efficiency. Moreover, glycosylation significantly boosted the thermostability of EGPf, allowing it to retain over 80% of its activity even after exposure to 100 °C for 5 h, with the optimal temperature being above 120 °C. Glycosylation did not affect the pH stability or salt tolerance of EGPf, although the glycosylated compound exhibited a high tolerance to ionic liquids. EGPf displayed the highest specific activity in the presence of 20% (v/v) 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), reaching approximately 2.4 times greater activity than that in the absence of [Bmim]Cl. The specific activity was comparable to that without the ionic liquid even in the presence of 40% (v/v) [Bmim]Cl. Glycosylated EGPf has potential as an enzyme for saccharifying cellulose under high-temperature conditions or with ionic liquid treatment due to its exceptional thermostability and ionic liquid tolerance. These results underscore the potential of N-glycosylation as an effective strategy to further enhance both the thermostability of highly thermostable archaeal enzymes and the hydrolysis of barley cellulose in the presence of [Bmim]Cl.

Establishment of a simple, sensitive, and specific ASFV detection method based on Pyrococcus furiosus argonaute.

African swine fever (ASF), which is casued by African swine fever virus (ASFV), is a fatal infectious disease of pigs that results in significant losses to the breeding industry. Therefore, screening and detection are crucial for the control and prevention of the ASFV. Argonaute is a new detection tool that is being extensively used due to its high specificity and programmability. This study reports on a new nucleic acid assay method, termed REPD, which uses recombinase-aided amplification and restriction endonuclease-assisted Pyrococcus furiosus argonaute (PfAgo) detection. One-pot REPD was developed for the detection of ASFV. The one-pot REPD could detect a single copy of ASFV nucleic acid and showed no cross-reactivity with other pathogens. Detection in clinical samples was 100% consistent with the results of real-time PCR analysis. The results showed that the one-pot REPD assay is convenient, sensitive, specific, and potentially adaptable to the detection of ASFV. In summary, this study highlights a novel method that can be employed for the detection of pathogens.

Pyrococcus furiosus argonaute based Alicyclobacillus acidoterrestrsis detection in fruit juice.

Alicyclobacillus acidoterrestris is the major threat to fruit juice for its off-odor producing characteristic. In this study, Pyrococcus furiosus Argonaute (PfAgo), a novel endonuclease with precise DNA cleavage activity, was used for A. acidoterrestrisdetection, termed as PAD. The partially amplified 16 S rRNA gene of A. acidoterrestris can be cleaved by PfAgo activated by a short 5'-phosphorylated single strand DNA, producing a new guide DNA (gDNA). Then, PfAgo was activated by the new gDNA to cut a molecular beacon (MB) with fluorophore-quencher reporter, resulting in the recovery of fluorescence. The fluorescent intensity is positively related with the concentration of A. acidoterrestris. The PAD assay showed excellent specificity and sensitivity as low as 101 CFU/mL, which can be a powerful tool for on-site detection of A. acidoterrestris in fruit juice industry in the future, reducing the economic loss.

Homo-trimeric structure of the ribonuclease for rRNA processing, FAU-1, from Pyrococcus furiosus.

Crystal structure of a ribonuclease for ribosomal RNA processing, FAU-1, from Pyrococcus furiosus was determined with the resolution of 2.57 Å in a homo-trimeric form. The monomer structure consists of two domains: N-terminal and C-terminal domains. C-terminal domain forms trimer and each N-terminal domain locates outside of the trimer core. In the obtained crystal, a dinucleotide, pApUp, was bound to the N-terminal domain, indicating that N-terminal domain has the RNA-binding ability. The affinities to RNA of FAU-1 and a fragment corresponding to the N-terminal domain, FAU-ΔC, were confirmed by polyacrylamide gel electrophoresis and nuclear magnetic resonance (NMR). Interestingly, well-dispersed NMR signals were observed at 318K, indicating that the FAU-ΔC-F18 complex form an ordered structure at higher temperature. As predicted in our previous works, FAU-1 and ribonuclease (RNase) E show a structural similarity in their RNA-binding regions. However, structural similarity between RNase E and FAU-1 could be found in the limited regions of the N-terminal domain. On the other hand, structural similarity between C-terminal domain and some proteins including a phosphatase was found. Thus, it is possible that the catalytic site is located in C-terminal domain.

Pyrococcus furiosus Argonaute-mediated porcine epidemic diarrhea virus detection.

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, induces severe vomiting and acute watery diarrhea in unweaned piglets. The pig industry has suffered tremendous financial losses due to the high mortality rate of piglets caused by PEDV. Consequently, a simple and rapid on-site diagnostic technology is crucial for preventing and controlling PEDV. This study established a detection method for PEDV using recombinase-aided amplification (RAA) and Pyrococcus furiosus Argonaute (PfAgo), which can detect 100 copies of PEDV without cross-reactivity with other pathogens. The entire reaction of RAA and PfAgo to detect PEDV does not require sophisticated instruments, and the reaction results can be observed with the naked eye. Overall, this integrated RAA-PfAgo cleavage assay is a practical tool for accurately and quickly detecting PEDV. KEY POINTS: • PfAgo has the potential to serve as a viable molecular diagnostic tool for the detection and diagnosis of viral genomes • The RAA-PfAgo detection technique has a remarkable level of sensitivity and specificity • The RAA-PfAgo detection system can identify PEDV without needing advanced equipment.

Enzyme-Assisted Endogenous Guide DNA Generation-Mediated Pyrococcus furiosus Argonaute for Alicyclobacillus acidoterrestris Detection.

Pyrococcus furiosusArgonaute (PfAgo) emerged as a novel endonuclease for the nucleic acid test recently. However, the input of exogenous guide DNA (gDNA) to activate PfAgo has reduced its flexibility. In this work, an enzyme-assisted endogenous gDNA generation-mediated PfAgo for the target detection strategy, termed EGG-PAD, was proposed. With the aid of EcoR Ι, the target double-strand DNA was cut, producing a phosphate group at the 5' end, functioning as gDNA to activate PfAgo for nucleic acid detection. The applicability of this assay was tested in the detection ofAlicyclobacillus acidoterrestris, a bacterium causing the spoilage of fruit juice, showing excellent sensitivity and specificity, ascribed to the "duplex amplification and triple insurance" mechanism. Moreover, EGG-PAD exhibited superior versatility in the identification of common foodborne pathogens. This powerful platform could also be an on-site test tool for detecting nucleic acid-containing organisms such as tumor cell, pathogen, and virus in the future.

Rapid and sensitive detection of Mycoplasma synoviae using RPA combined with Pyrococcus furiosus Argonaute.

Mycoplasma synoviae (MS) is an important pathogen in laying hens and causes serious economic losses in poultry production. Rapid, accurate and specific detection is important for the prevention and control of MS. Argonaute from Pyrococcus furiosus (PfAgo) is emerging as a nucleic acid detector that works via "dual-step" sequence-specific cleavage. In this study, an MS detection method combining recombinase polymerase amplification (RPA) and PfAgo was established. Through elaborate design and screening of RPA primers and PfAgo gDNA and condition optimization, amplification and detection procedures can be completed within 40 min, whereas the results were superficially interpreted under UV and blue light. The sensitivity for MS detection was 2 copies/µL, and the specificity results showed no cross reaction with other pathogens. For the detection of 31 clinical samples, the results of this method and qPCR were completely consistent. This method provides a reliable and convenient method for the on-site detection of MS that is easy to operate without complex instruments and equipment.

Molecular mechanism for target recognition, dimerization, and activation of Pyrococcus furiosus Argonaute.

The Argonaute nuclease from the thermophilic archaeon Pyrococcus furiosus (PfAgo) contributes to host defense and represents a promising biotechnology tool. Here, we report the structure of a PfAgo-guide DNA-target DNA ternary complex at the cleavage-compatible state. The ternary complex is predominantly dimerized, and the dimerization is solely mediated by PfAgo at PIWI-MID, PIWI-PIWI, and PAZ-N interfaces. Additionally, PfAgo accommodates a short 14-bp guide-target DNA duplex with a wedge-type N domain and specifically recognizes 5'-phosphorylated guide DNA. In contrast, the PfAgo-guide DNA binary complex is monomeric, and the engagement of target DNA with 14-bp complementarity induces sufficient dimerization and activation of PfAgo, accompanied by movement of PAZ and N domains. A closely related Argonaute from Thermococcus thioreducens adopts a similar dimerization configuration with an additional zinc finger formed at the dimerization interface. Dimerization of both Argonautes stabilizes the catalytic loops, highlighting the important role of Argonaute dimerization in the activation and target cleavage.

Crystal structure of a thiolase from Archaeal Pyrococcus furiosus and its in silico functional annotation.

In most of the eukaryotes and archaea, isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP) essential building blocks of all isoprenoids synthesized in the mevalonate pathway. Here, the first enzyme of this pathway, acetoacetyl CoA thiolase (PFC_04095) from an archaea Pyrococcus furiosus is structurally characterized. The crystal structure of PFC_04095 is determined at 2.7 Å resolution, and the crystal structure reveals the absence of catalytic acid/base cysteine in its active site, which is uncommon in thiolases. In place of cysteine, His285 of HDAF motif performs both protonation and abstraction of proton during the reaction. The crystal structure shows that the distance between Cys83 and His335 is 5.4 Å. So, His335 could not abstract a proton from nucleophilic cysteine (Cys83), resulting in the loss of enzymatic activity of PFC_04095. MD simulations of the docked PFC_04095-acetyl CoA complex show substrate binding instability to the active site pocket. Here, we have reported that the stable binding of acetyl CoA to the PFC_04095 pocket requires the involvement of three protein complexes, i.e., thiolase (PFC_04095), DUF35 (PFC_04100), and HMGCS (PFC_04090).

RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses.

The advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. In this study, a novel detection system was developed by integrating reverse transcriptome (RT) techniques with recombinase polymerase isothermal amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo). The RT-RPA-PfAgo system enabled the simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV). Identifying targets via guide DNA without being hindered by protospacer adjacent motif sequences is the inherent merit of PfAgo, with the additional advantage of it being simple, cost-effective, and exceptionally sensitive, with detection limits between 3.13 and 5.13 copies/µL, in addition to it effectively differentiating between the three distinct viruses. The field evaluations were also in accordance with RT-PCR methods. The RT-RPA-PfAgo system proved to be a robust, versatile, highly specific, and sensitive method with great potential for practicality in future plant virus diagnostics.

SARS-CoV-2 Spike protein peptides displayed in the Pyrococcus furiosus RAD system preserve epitopes antigenicity, immunogenicity, and virus-neutralizing activity of antibodies.

Amongst the potential contribution of protein or peptide-display systems to study epitopes with relevant immunological features, the RAD display system stands out as a highly stable scaffold protein that allows the presentation of constrained target peptides. Here, we employed the RAD display system to present peptides derived from the SARS-CoV-2 Spike (S) protein as a tool to detect specific serum antibodies and to generate polyclonal antibodies capable of inhibiting SARS-CoV-2 infectivity in vitro. 44 linear S-derived peptides were genetically fused with the RAD scaffold (RAD-SCoV-epitopes) and screened for antigenicity with sera collected from COVID-19-infected patients. In a second step, selected RAD-SCoV-epitopes were used to immunize mice and generate antibodies. Phenotypic screening showed that some of these antibodies were able to recognize replicating viral particles in VERO CCL-81 and most notably seven of the RAD-SCoV-epitopes were able to induce antibodies that inhibited viral infection. Our findings highlight the RAD display system as an useful platform for the immunological characterization of peptides and a potentially valuable strategy for the design of antigens for peptide-based vaccines, for epitope-specific antibody mapping, and for the development of antibodies for diagnostic and therapeutic purposes.

The archaeal Lsm protein from Pyrococcus furiosus binds co-transcriptionally to poly(U)-rich target RNAs.

Posttranscriptional processes in Bacteria include the association of small regulatory RNAs (sRNA) with a target mRNA. The sRNA/mRNA annealing process is often mediated by an RNA chaperone called Hfq. The functional role of bacterial and eukaryotic Lsm proteins is partially understood, whereas knowledge about archaeal Lsm proteins is scarce. Here, we used the genetically tractable archaeal hyperthermophile Pyrococcus furiosus to identify the protein interaction partners of the archaeal Sm-like proteins (PfuSmAP1) using mass spectrometry and performed a transcriptome-wide binding site analysis of PfuSmAP1. Most of the protein interaction partners we found are part of the RNA homoeostasis network in Archaea including ribosomal proteins, the exosome, RNA-modifying enzymes, but also RNA polymerase subunits, and transcription factors. We show that PfuSmAP1 preferentially binds messenger RNAs and antisense RNAs recognizing a gapped poly(U) sequence with high affinity. Furthermore, we found that SmAP1 co-transcriptionally associates with target RNAs. Our study reveals that in contrast to bacterial Hfq, PfuSmAP1 does not affect the transcriptional activity or the pausing behaviour of archaeal RNA polymerases. We propose that PfuSmAP1 recruits antisense RNAs to target mRNAs and thereby executes its putative regulatory function on the posttranscriptional level.