Proton conductors, particularly hydrated solid membranes, have various applications in sensors, fuel cells, and cellular biological systems. Unraveling the intrinsic proton transfer mechanism is critical for establishing the foundation of proton conduction. Two scenarios on electrical conduction, the Grotthuss and the vehicle mechanisms, have been reported by experiments and simulations. But separating and quantifying the contributions of these two components from experiments is difficult. Here, we present the conductive behavior of a two-dimensional layered proton conductor, graphene oxide membrane (GOM), and find that proton hopping is dominant at low water content, while ion diffusion prevails with increasing water content. This change in the conduction mechanism is attributable to the layers of water molecules in GOM nanosheets. The overall conductivity is greatly improved by forming one layer of water molecules. It reaches the maximum with two layers of water molecules, resulting from creating a complete hydrogen-bond network within GOM. When more than two layers of water molecules enter the GOM nanosheets, inducing the breakage of the ordered lamellar structure, protons spread in both in-plane and out-of-plane directions inside the GOM. Our results validate the existence of two conduction mechanisms and show their distinct contributions to the overall conductivity. Furthermore, these findings provide an optimization strategy for the design of realizing the fast proton transfer in materials with water participation.

Conduction Mechanism in Graphene Oxide Membranes with Varied Water Content: From Proton Hopping Dominant to Ion Diffusion Dominant.

Prokaryotic Argonautes (pAgos) have been recently used in many nucleic acid biosensing applications but have rarely been used for regulating the isothermal amplification system. Herein, we reported Thermus thermophilus Argonaute (TtAgo)-mediated background-suppressed exponential isothermal amplification (EXPAR) as the first example to explore the binding activity of pAgos toward regulation of the amplification template. It was demonstrated that thermophilic pAgos efficiently eliminated nonspecific hybridization between templates by their binding affinity with the template, resulting in greatly enhancing the specificity of EXPAR. TtAgo-mediated, background-suppressed EXPAR was employed to detect miRNA with a detection limit of 10-15 M, which was 1000 times and 100 times more sensitive than that of traditional RT-PCR and EXPAR, respectively. This method further showed good performance in discriminating cancer patients from healthy individuals, indicating its potential for practical clinical applications.

Binding Activity of Prokaryotic Argonaute for Background-Suppressed Exponential Isothermal Amplification.

Foodborne pathogens endanger human health. Therefore, sensitive and accurate detection assays are key for their prevention and control. Microdroplet digital nucleic acid platforms can achieve single-molecule sensitivity in the presence of specialized instrumentation for generating homogenized monodisperse droplets, expensive thermal cyclers, high temperature-resistant reagents, and time-consuming multistep operations in the lab. Argonaute (Ago), an emerging next-generation gene editing tool, can be used for the detection of pathogen genetic information. Current reports mainly adopt the high temperature-dependent thermophilic Ago in stepwise nucleic acid detection, which hinders its applications in ultrasensitive digital detection. Here, we expressed and purified the mesophilic Clostridium butyricum Argonaute (CbAgo) and developed a polydisperse microdroplet reactors-assist asymmetric recombinase polymerase amplification driven CbAgo report digital detection platform (DRAGON). It realized the signal-enhanced nucleic acid amplification and CbAgo report compatibility in novel low-cost and ultrafast-prepared polydisperse microdroplet reactors. The microdroplet reactor area and fluorescence intensity were combined to establish a dual parameter-based random forest machine learning model, which achieved high sensitivity (1 CFU/mL) and efficiency (<45 min) for detecting pathogens. This is the first time that mesophilic Ago was used to achieve true sensitive one-step and one-pot low-temperature digital nucleic acid detection. Our system accomplished multiplex detection in one-step and one-pot with a single nuclease which is impossible for CRISPR/Cas system and thermophilic Ago-based stepwise platforms. DRAGON represents the next-generation platform for pathogen detection based on gene editing tools and intelligent digital nucleic acid detection system. 

A digital platform for One-Pot signal enhanced foodborne pathogen detection based on mesophilic argonaute-driven polydisperse microdroplet reactors and machine learning

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. 

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

Isothermal amplification method has enabled major advancement toward rapid and deployable detection for nucleic acid. While exciting, there are still a series of deficiencies facing their practical implementation, such as carryover cross-contamination, which are major bottlenecks that must be addressed. In addition, there is a lack of high-throughput detection for foodborne pathogens in foods. Here, we reported an Argonaute-mediated bio-barcode bioassay for one-step cleavage, on-site and high-throughput detection of S. aureus (Staphylococcus aureus) through introducing Tag sequence and recombinase aided amplification (RAA). Furthermore, we designed a single vessel via tube-in-tube manner for one-tube contamination-free and portable detection without opening the lid. With this strategy, the same detection limit as fluorescence detection could be obtained, 1 CFU/mL with high specificity for S. aureus. We further evaluated the performance of bio-barcode biosensing strategy using S. aureus-contaminated clinical and food samples. Argonaute-mediated bio-barcode bioassay had the advantages of modular, universal plug-and-play and high-throughput scanning. This work not only satisfied the requirement for convenient detection but also fulfilled the need for portable on-site nucleic acid detection with portability, adaptability, transferability and practicality. 

An Argonaute-mediated bio-barcode bioassay for one-tube and on-site detection of Staphylococcus aureus

Abstract Prokaryotic Argonaute proteins (pAgos) widely participate in hosts to defend against the invasion of nucleic acids. Compared with the CRISPR-Cas system, which requires a specific motif on the target and can only use RNA as guide, pAgos exhibit precise endonuclease activity on any arbitrary target sequence and can use both RNA and DNA as guide, thus rendering great potential for genome editing applications. Hitherto, most in-depth studies on the structure-function relationship of pAgos were conducted on thermophilic ones, functioning at ∼60 to 100°C, whose structures were, however, determined experimentally at much lower temperatures (20–33°C). It remains unclear whether these low-temperature structures can represent the true conformations of the thermophilic pAgos under their physiological conditions. The present work studied three pAgos, PfAgo, TtAgo and CbAgo, whose physiological temperatures differ significantly (95, 75 and 37°C). By conducting thorough experimental and simulation studies, we found that thermophilic pAgos (PfAgo and TtAgo) adopt a loosely-packed structure with a partially-melted surface at the physiological temperatures, largely different from the compact crystalline structures determined at moderate temperatures. In contrast, the mesophilic pAgo (CbAgo) assumes a compact crystalline structure at its optimal function temperature. Such a partially-disrupted structure endows thermophilic pAgos with great flexibility both globally and locally at the catalytic sites, which is crucial for them to achieve high DNA-cleavage activity. To further prove this, we incubated thermophilic pAgos with urea to purposely disrupt their structures, and the resulting cleavage activity was significantly enhanced below the physiological temperature, even at human body temperature. Further testing of many thermophilic Agos present in various thermophilic prokaryotes demonstrated that their structures are generally disrupted under physiological conditions. Therefore, our findings suggest that the highly dynamical structure with a partially-melted surface, distinct from the low-temperature crystalline structure, could be a general strategy assumed by thermophilic pAgos to achieve the high DNA-cleavage activity.

/pdf/loosely-packed-dynamical-structures-with-partially-melted-1t34tlwz.pdf

Loosely-packed dynamical structures with partially-melted surface being the key for thermophilic argonaute proteins achieving high DNA-cleavage activity

Interfacial water remains liquid and mobile much below 0 °C, imparting flexibility to the encapsulated materials to ensure their diverse functions at subzero temperatures. However, a united picture that can describe the dynamical differences of interfacial water on different materials and its role in imparting system-specific flexibility to distinct materials is lacking. By combining neutron spectroscopy and isotope labeling, we explored the dynamics of water and the underlying substrates independently below 0 °C across a broad range of materials. Surprisingly, while the function-related anharmonic dynamical onset in the materials exhibits diverse activation temperatures, the surface water presents a universal onset at a common temperature. Further analysis of the neutron experiment and simulation results revealed that the universal onset of water results from an intrinsic surface-independent relaxation: switching of hydrogen bonds between neighboring water molecules with a common energy barrier of ∼35 kJ mol−1.

/pdf/universal-dynamical-onset-in-water-at-distinct-material-3ezszrv9.pdf

Universal dynamical onset in water at distinct material interfaces

Membrane‐active peptides play an essential role in many living organisms and their immune systems and counter many infectious diseases. Many have dual or multiple mechanisms and can synergize with other molecules, like peptides, proteins, and small molecules. Although membrane‐active peptides have been intensively studied in the past decades and more than 3500 sequences have been identified, only a few received approvals from the US Food and Drug Administration. In this review, we investigated all the peptide therapeutics that have entered the market or were subjected to preclinical and clinical studies to understand how they succeeded. With technological advancement (e.g., chemical modifications and pharmaceutical formulations) and a better understanding of the mechanism of action and the potential targets, we found at least five membrane‐active peptide drugs that have entered preclinical/clinical phases and show promising results for cancer treatment. We summarized our findings in this review and provided insights into membrane‐active anticancer peptide therapeutics.

Development of membrane‐active peptide therapeutics in oncology

Prokaryotic Argonaute (pAgo) proteins, a class of DNA/RNA-guided programmable endonucleases, have been extensively utilized in nucleic acid-based biosensors. The specific binding and cleavage of nucleic acids by pAgo proteins, which...

Mn2+-Induced Structural Flexibility Enhances the Entire Catalytic Cycle and the Cleavage of Mismatches in Prokaryotic Argonaute Proteins

Prokaryotic Argonaute (pAgo) proteins, a class of DNA/RNA-guided programmable endonucleases, have been extensively utilized in nucleic acid biosensors. The specific binding and cleavage of nucleic acids by pAgo proteins, which are crucial processes for their applications, are dependent on the presence of Mn2+ bound in the pockets, as already verified through X-ray crystallography. However, a comprehensive understanding of how dissociated Mn2+ in the solvent affects the catalytic cycle, and its underlying regulatory role in this structure-function relationship, remains underdetermined. By combining experimental and computational methods, this study reveals that unbound Mn2+ significantly enhances the flexibility of diverse pAgo proteins, regardless of their classification as hyperthermophiles, thermophiles, or mesophiles. This increase in flexibility through decreasing the number of hydrogen bonds, induced by Mn2+, leads to higher affinity for substrates, thus facilitating cleavage. More importantly, Mn2+-induced structural flexibility increases the mismatch tolerance between guide-target pairs by increasing the conformational states, thereby enhancing the cleavage of mismatches. Further simulations indicated that the enhanced flexibility in linkers triggers conformational changes in the PAZ domain for recognizing various lengths of nucleic acids. Additionally, the Mn2+-induced dynamic alterations of the protein cause a conformational shift in the N domain and catalytic sites towards their functional form, resulting in a decreased energy penalty for target release and cleavage. These findings demonstrate that the dynamic conformations of pAgo proteins, resulting from the presence of the unbound Mn2+, significantly promote the catalytic cycle of endonucleases and the tolerance of cleavage to mismatches. This flexibility enhancement mechanism serves as a general strategy employed by Ago proteins from diverse prokaryotes to accomplish their catalytic functions and provide useful information for Ago-based precise molecular diagnostics.

/pdf/mn2-induced-structural-flexibility-enhances-the-entire-uxxp9jbg.pdf

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Loosely-packed dynamical structures with partially-melted surface being the key for thermophilic argonaute proteins achieving high DNA-cleavage activity

Universal dynamical onset in water at distinct material interfaces

Development of membrane‐active peptide therapeutics in oncology

Mn2+-Induced Structural Flexibility Enhances the Entire Catalytic Cycle and the Cleavage of Mismatches in Prokaryotic Argonaute Proteins

Mn2+-Induced Structural Flexibility Enhances the Entire Catalytic Cycle and the Cleavage of Mismatches in Prokaryotic Argonaute Proteins