Discriminating native, large protein assemblies from their oligomeric intermediates is inherently challenging as minor differences in size and shape often limit the resolution of routine analytical methods. Here, we present conformation-specific electrophoresis (CSE), a multistack gel strategy enabling quantitative, subunit-resolution separation of native complex oligomers in a simple slab-gel format. CSE employs tailored polyacrylamide stacks imposing iterative focusing and differential sieving, thereby sharpening and sorting analytes based on subtle differences in shape and mass. Using a pore-forming toxin as a model system, CSE fully resolved all structurally unique assembly intermediates, whereas conventional electrophoresis methods typically yielded only one or two overlapping bands. The method provides high resolution, analytical linearity, and sensitivity for quantifying trace oligomers. We further demonstrate its broad applicability for nondestructive gel excision to isolate functional oligomers, confirming the method's nondenaturing nature and providing material for subsequent advanced analysis, such as single-molecule nanopore techniques. By integrating high analytical performance and compatibility with standard electrophoresis equipment, CSE provides a practical platform for investigating assembly mechanisms and serving as an effective tool for characterizing molecular heterogeneity in macromolecular chemistry, structural biology, and pharmaceutical science.

Abrin, a potent ribosome-inactivating protein, is a serious threat as a biological warfare agent. Developing rapid and accurate assays for detecting abrin are crucial for early prevention of related attacks. In this study, we systematically demonstrate that abrin exhibits apurinic/apyrimidinic (AP) lyase activity, with optimal enzymatic function at 63 degrees C and pH 4.4. Based on this discovery, we create a fluorescence signaling assay (FSA) for swift detecting abrin and further integrate it with a lateral flow assay (LFA), resulting in a novel, portable detection platform (FSA-LFA). This combined assay allows for sensitive detection of abrin at the picogram level, surpassing the limitations of traditional N-glycosidase-based assays. The FSA-LFA platform is cost-effective, highly sensitive, and amenable to field use. Furthermore, it can be readily adapted for the detection of other ribosome-inactivating proteins, offering broad potential for rapid identification of biosecurity threats.

Ricin toxin (RT) is a potential bioterrorism agent because of its high potency, extremely small lethal dose, ease of preparation, and notable stability. Therefore, a portable method is urgently required to efficiently detect and determine the presence of toxicity of RT and evaluate its potency for public health monitoring and counter-bioterrorism responses. Currently, enzyme-based assays for detecting RT mainly focus on its N-glycosidase activity. In this study, we demonstrated that RT exhibits apurinic/apyrimidinic (AP) lyase activity using several methods. Characterization of the enzyme reaction and kinetics revealed that AP lyase activity is optimal at 59 °C and pH 4.0. This activity is highly pH-sensitive, remaining active between pH 3.0 and pH 4.6. Furthermore, we developed a portable fluorescence-based lateral flow assay (FLFA) that detects RT much faster than existing assays based on its N-glycosidase activity. Moreover, this assay can efficiently detect RT at nanogram levels from complex matrix samples within 1.5 h while simultaneously determining its biological activity. In conclusion, the discovery of the AP lyase activity of RT and the development of FLFA represent novel approaches for studying the enzymatic profiles of other ribosome-inactivating proteins.

DNA nanostructures have shown great potential in biomedical fields. However, the immune responses, especially the activation of the cGAS-STING signaling (A-cGSs), induced by DNA nanostructures, remain incompletely understood. Here, the ability of various DNA nanostructures from double-stranded DNA (dsDNA), single-stranded tiles (SSTs) to DNA origami is investigated on A-cGSs. Unlike natural dsDNA which triggers potent A-cGSs, the structural interconnectivity of various DNA configurations can substantially reduce the occurrence of A-cGSs, irrespective of their form, dimensions, and conformation. However, wireframe DNA nanostructures can activate the cGAS-STING signaling, suggesting that decreasing A-cGSs is dsDNA compactness-dependent. Based on this, a reconfigurable DNA Origami Domino Array (DODA) is used to systematically interrogate how dsDNA influences the A-cGSs and demonstrates that the length, number, and space of dsDNA array coordinately influence the activation level of cGAS-STING signaling, realizing a regulation of innate immune response. The above data and findings enhance the understanding of how DNA nanostructures affect cellular innate immune responses and new insights into the modulation of innate immune responses by DNA nanomedicine.

Clostridium perfringens alpha toxin (CPA), which causes yellow lamb disease in sheep and gas gangrene and food poisoning in humans, is produced by all types of C. perfringens and is the major virulence determinant of C. perfringens type A. CPA induces hemolysis in many species, including humans, murines, sheep and rabbits, through its enzymatic activity, which dissolves the cell membrane. Recent studies have shown that some pore-forming toxins cause hemolysis, which is achieved by the activation of purinergic receptors (P2). However, the relationship between P2 receptors and non-pore-forming toxin hemolysis has not been investigated. In the present study, we examined the function of P2 receptors in CPA toxin hemolysis and found that CPA-induced hemolysis was dependent on P2 receptor activation, and this was also true for Staphylococcus aureus β-Hemolysin, another non-pore-forming toxin. Furthermore, we use selective P2 receptor antagonists to demonstrate that P2X1 and P2X7 play important roles in the hemolysis of human and murine erythrocytes. In addition, we found that redox metabolism was mainly involved in CPA-induced hemolysis using metabolomic analysis. We further demonstrate that CPA activates P2 receptors and then activates NADPH oxidase through the PI3K/Akt and MEK1/ERK1 pathways, followed by the production of active oxygen to induce hemolysis. These findings contribute to our understanding of the pathological effects of CPA, clarify the relationship between P2 activation and non-pore-forming toxin-induced hemolysis, and provide new insights into CPA-induced hemolysis.

Hemolysis is the process of rupturing erythrocytes (red blood cells) by forming nanopores on their membranes using hemolysins, which then impede membrane permeability. However, the self-assembly process before the state of transmembrane pores and underlying mechanisms of conformational change are not fully understood. In this work, theoretical and experimental evidence of the pre-pore morphology of Clostridium perfringens epsilon toxin (ETX), a typical hemolysin, is provided using in situ atomic force microscopy (AFM) complemented by molecular dynamics (MD) simulations to detect the conformational distribution of different states in Mica. The AFM suggests that the ETX pore is formed in two stages: ETX monomers first attach to the membrane and form a pre-pore in no special conditions required, which then undergo a conformational change to form a transmembrane pore at temperatures above the critical point in the presence of receptors. The authors' MD simulations reveal that initial nucleation occurs when specific amino acids adsorb to negatively charged mica cavities. This work fills the knowledge gap in understanding the early stage of hemolysis and the oligomerization of hemolysins. Moreover, the newly identified pre-pore of ETX holds promise as a candidate for nanopore applications.

Inspired by efficient biomolecular reactions in the cell, versatile DNA nanostructures have been explored for manipulating the spatial position and regulating reactions at the molecular level. Spatially controlled arrangement of molecules on the artificial scaffolds generally leads to enhanced reaction activities. Especially, the rich toolset of dynamic DNA nanostructures provides a potential route towards more sophisticated and vigorous regulation of molecular reactions. Herein, a reconfigurable DNA origami domino array (DODA) as a dynamic scaffold was adopted in this work for temporal-controlled and switchable molecular cascade reactions. Dynamic regulation of the assembly of G-quadruplex, hybridization of parallel-stranded duplex and assembly of binary DNAzyme were demonstrated. Molecular cascade reactions on the triggered reconfiguration of DODAs were realized, resulting in more complex, dynamic, and switchable control over the reactions.

Structural DNA nanotechnology has been applied to construct complex static and dynamic DNA structures. Seeded growth, intended to regulate the crucial nucleation step, has been used to control the assembly of DNA tiles. However, most of the seeded growth strategies were applied to fixed DNA tiles, whereas the seeded growth on dynamic DNA tiles remains unreported. Here, we propose a seeded growth strategy in which dynamic tiles adaptively change their shapes to match the architecture of a DNA origami seed. Furthermore, when more adaptive DNA tiles assemble on a reconfigurable DNA origami domino array (DODA) seed, the conformation of DODA seeds is reversibly affected by the spontaneous reconfiguration of adaptive tiles. The adaptive seeded growth provides a mechanism for the construction of complex DNA nanomachines and may offer a general and adaptable method for the advancement of responsive materials, with active, autonomous, and adaptive spatiotemporal control properties.

Despite its polyanionic nature, DNA can cross the negatively charged membrane to enter living cells by assembling into specific nanostructures, establishing various opportunities for biomedical applications. Mechanistic studies to explain how the geometrical parameters of DNA nanostructures impact the cell entry are critical but elusive. Here, we use experimentation and simulation to study the interaction between cells and three typical framework nucleic acids (FNAs), including tetrahedron, triangular prism, and cube. Different cellular uptake efficiency was observed among these FNAs, and similar distinction consistently existed in multiple cell lines. Scavenger receptors (SRs) were demonstrated to be essential in mediating the uptake process. Molecular docking simulations revealed that the SR binding predominantly depended on the corner angle of FNAs, determining cellular internalization frequency. This study clearly explains how FNAs interact with the membrane to initiate cell entry, offering new clues for the design of theranostic nanocarriers and the study of virus invasion.

The community-associated Methicillin-resistant Staphylococcus aureus strain (CA-MRSA) is highly virulent and has become a major focus of public health professionals. Phenol-soluble modulins (PSM) are key factors in its increased virulence. δ-Toxin belongs to PSM family and has copious secretion in many S. aureus strains. In addition, δ-toxin exists in the S. aureus culture supernatant as both N-terminus formylated δ-toxin (fδ-toxin) and deformylated δ-toxin (dfδ-toxin) groups. Although δ-toxin has been studied for more than 70 years, its functions remain unclear. We isolated and purified PSMs from the supernatant of S. aureus MW2, and found fibrils and oligomers aggregates by Size Exclusion Chromatography. After analyzing PSM aggregates and using peptide simulations, we found that the difference in the monomer structure of fδ-toxin and dfδ-toxin might ultimately lead to differences in the aggregation ability: fδ-toxin and dfδ-toxin tend to form fibrils and oligomers respectively. Of note, we found that fδ-toxin fibrils enhanced the stability of biofilms, while dfδ-toxin oligomers promoted their dispersal. Additionally, oligomeric dfδ-toxin combined with PSMα to form a complex with enhanced functionality. Due to the different aggregation capabilities and functions of fδ-toxin and dfδ-toxin, we speculate that they may be involved in the regulation of physiological activities of S. aureus. Moreover, the dfδ-toxin oligomer not only provides a new form of complex in the study of PSMα, but also has significance as a reference in oligomer research pertaining to some human amyloid diseases.

DNA origami has represented a novel route to manipulate objects at nanoscale, and demonstrated unprecedented versatility in fabricating both static and dynamic nanostructures. Here, we introduce a new strategy for transferring modular reconfigurable DNA nanostructures from 2D to 3D. A 2D DNA sheet could be modularized into connected parts (e.g. two, three and four parts in this work), which can be independently transformed between two conformations with a few DNA "trigger" strands. More interestingly, the transformation of the connected 2D modules can lead to the controlled, resettable structural conversion of a 2D sheet to a 3D architecture, due to the constraints induced by the connections between the 2D modules. This new approach can provide an efficient mean for constructing programmable, higher-order, and complex DNA objects, as well as sophisticated dynamic substrates for various applications.

The transdermal drug delivery approach has been considered a potential therapy for human hypertrophic scars (HSs) instead of current uncomfortable surgical excision, local injection and laser therapy. However, a facile and efficient drug delivery method is urgently needed to overcome the skin barrier of transdermal administration. Herein, we employed a DNA-Fe nanoparticle delivery systemviaFe ion driven self-assembly to satisfy the requirement of transdermal administration for HS therapy. Doxorubicin hydrochloride (DOX) as one of the widely used anticancer drugs was employed to treat the hyperplasia of abnormal skin fibrous tissue. Bothin vitroandin vivoexperiments of the DOX loaded DNA-Fe nanoparticles (DOX@DNA-Fe NPs) were performed to demonstrate the penetration ability, rapid drug release, and scar-inhibiting effects. This facile and efficient approach for HS therapyviaa DNA-based transdermal drug delivery system may provide more possibilities for the development of transdermal administration.

DNA origami enables the manipulation of objects at nanoscale, and demonstrates unprecedented versatility for fabricating both static and dynamic nanostructures. In this work, we introduce a new strategy for transferring modular reconfigurable DNA nanostructures from two-dimensional to three-dimensional. A 2D DNA sheet could be modularized into connected parts (e.g., two, three, and four parts in this work), which can be independently transformed between two conformations with a few DNA "trigger" strands. More interestingly, the transformation of the connected 2D modules can lead to the controlled, resettable structural conversion of a 2D sheet to a 3D architecture, due to the constraints induced by the connections between the 2D modules. This new approach can provide an efficient mean for constructing programmable, higher-order, and complex DNA objects, as well as sophisticated dynamic substrates for various applications.

The DNA origami technique is a robust method for the design of DNA nanostructures with prescribed shapes, including complex curved geometries. In addition to static structures, dynamic DNA origami has been used to construct sophisticated nanomachines that can reconfigure their shapes in response to external stimuli. Here, we report a new method to design DNA origami structures that can transform between a noncurved conformation and curved conformation. The reconfigurable structures are developed on the basis of dynamic DNA domino origami, which can transform in a cascading process initiated by trigger DNA strands. The degree of curvature could be programmed by tuning the sizes of DNA units within the origami.

Catalytic DNA molecules (DNAzymes), produced through the systematic evolution of ligands by exponential enrichment (SELEX) process, are synthetic, single-stranded DNA molecules that either have catalytic abilities or can perform specific reactions. Among these DNAzymes, RNA-cleaving DNAzymes are the ones that can cleave RNAs at specific sites with the help of cofactors. The cofactors contains heavy metal ions (eg., Pb2+, Mg2+, Cu2+), small molecules (eg., ATP, L-histidine, Vc), bacteria (eg., Escherichia coli) and so on. Based on the particular property, the RNA-cleaving DNAzymes are particularly promising for creating methods that can detect a wide variety of targets. For example, scientists have successfully found the Pb2+ DNAzyme, Cu2+ DNAzyme, UO22+ DNAzyme and some other specific metal ions.based DNAzymes. These DNAzymes have a high recognition specificity for the metal ions. Only when the specific metal ions existed, can the catalytic activity be performed. Besides, and the size of the catalytic activity is closely related to the concentration of metal ions. Therefore, these DNAzymes can be used to detect heavy metal ions. Similarly, the DNAzymes have been used to detect the ATP, L-histidine and Escherichia coli. What should be mentioned is that natural DNAzymes are generally D-type nucleic acids, which can be easily degraded by proteinase in physiological fluid. Therefore, in order to extend the applications of DNAzymes, emphasis has been placed on improving the selectivity and stability of DNAzymes. Based on the principle of enantiomer of nucleic acid, non-natural L-type nucleic acids have been used to prepare DNAzymes. L-type DNAzymes have similar thermal stability to D-DNAzymes, they also have better biostability and are ideal materials for constructing biosensors for complex system detection. Furthermore, based on the property of cleaving RNAs at specific sites, the DNAzymes can not only be used to detection, but also can be used to inactivate target cellular mRNA, which can be further applied in the treatment of multiple clinical disease. However, in fact, the gene therapy of DNAzymes in tumor and pathogenic microorganisms is only active in the scientific research stage, there is still a long way to realize the real clinical applications. The most prominent problem is the delivery problems, that is, how to choose a safe, efficient and specific guiding carrier to deliver DNAzyme to the target gene. In recent years, the rise of nanomedicine has brought new opportunities for gene therapy. The use of nano-sized materials to construct the drug delivery system can effectively deliver genetic drugs to tumor tissues. Nano drug carrier is an effective means to improve drug bioavailability, enhance drug stability and improve drug targeted therapy. In this review, we summarized the researches on DNAzyme-based metal ion sensors and gene treatment, and in the basis, we also outlook the possibility that whether the DNAzymes can be efficiently used to specifically detect the targets in vivo, as well as their applications of diseases therapy.

Epsilon-toxin (ETX) is produced by types B and D strains of Clostridium perfringens, which cause fatal enterotoxaemia in sheep, goats and cattle. Previous studies showed that only a restricted number of cell lines are sensitive to ETX and ETX-induced hemolysis has not previously been reported. In this study, the hemolytic ability of ETX was examined using erythrocytes from 10 species including murine, rabbit, sheep, monkey and human. We found that ETX caused hemolysis in human erythrocytes (HC50 = 0.2 μM) but not erythrocytes from the other test species. Moreover, the mechanism of ETX-induced hemolysis was further explored. Recent studies showed that some bacterial toxins induce hemolysis through purinergic receptor (P2) activation. Hence, the function of purinergic receptors in ETX-induced hemolysis was tested, and we found that the non-selective P2 receptor antagonists PPADS inhibited ETX-induced lysis of human erythrocytes in a concentration-dependent manner, indicating that ETX-induced hemolysis requires activation of purinergic receptors. P2 receptors comprise seven P2X (P2X1–7) and eight P2Y (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11–P2Y14) receptor subtypes. The pattern of responsiveness to more selective P2-antagonists implies that both P2Y13 and P2X7 receptors are involved in ETX-induced hemolysis in human species. Furthermore, we demonstrated that extracellular ATP is likely not involved in ETX-induced hemolysis and the activation of P2 receptors. These findings clarified the mechanism of ETX-induced hemolysis and provided new insight into the activities and ETX mode of action.

Studying the self-assembly behavior of DNA origami allows a better understanding of molecular assembly characteristics at the nanoscale. Presently, the mechanisms governing growth and dynamics of DNA origami assembly are still not very clear and there is a lack of direct visualization of the growth processes on the long single-strand scaffold. Here, we investigate the kinetics, especially the real-time seeding growth process of six special designs of 2D DNA origami at room temperature (RT) without the assistance of denaturing chemicals. The prealignment of single-strand long scaffold and logical seeding growth behaviors are revealed during the growth process at RT. Furthermore, we studied the thermal stability of the DNA nanostructures under limited structural defects. Revealed characteristics of seeding growth can be used to build large and complex DNA nanodevices capable of performing logical operations with nanometer precision.

Diffusion plays a critical role in establishing functional bio/solid soft interfaces for bioassays, biosensors, and biofuel cells. An understanding of micro-diffusion near the interface is significant for developing high-performance bioassays, biosensors, and biofuel cells. Herein, we explored micro-diffusion behavior at different temperature gaps in microfluidic chip and enzyme-linked immunosorbent assay (ELISA) microplates. It exhibited that temperature gap could distinctly promote directional diffusion of molecules in microfluidic chip from high concentration zone to low concentration zone. In addition, experimental results of specially designed ELISA also partly confirmed that the temperature gap could effectively improve the performance of ELISA by 54.5%. In compared with conventional ELISA, the as-prepared temperature-gap functional module in microfluidic chip has obvious advantages of miniaturization, integration, customization, and low cost. All in all, the current work indicates that temperature-gap function has great potential in biomedical detection, food safety, and so on. It can be utilized to develop novel biological detection approaches and related instruments or microfluidic chips.

Infant botulism was rarely reported in China. The second reported event of the disease including three cases occurred in 2015. In the present study, one (the third case) of the three cases was identified and investigated to trace the sources of transmission. Samples from feces and foodstuffs were used to isolate Clostridium botulinum strains. Each isolate was obtained from the baby's feces and opened powdered infant rice cereal, respectively. In this case, the C. botulinum strains were identified and characterized by combined mouse bioassay, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high-throughput sequencing including single nucleotide polymorphisms (SNP). Results showed that the disease was caused by a type B strain of C. botulinum. Strains associated with this case as well as isolates from stored and historical samples were phylogenetically analyzed and compared. C. botulinum type B isolates from the infant feces and from an opened container of infant rice cereal were indistinguishable, suggesting that opened container of infant rice cereal is likely to be the source of transmission of spores to the infant. It is not clear that how the opened container was contaminated and the child was exposed since environmental testing was not performed. This study provides detailed information about usage of the three methods and references for dealing with other associated cases.

There is an increasing urge to investigate facile solutions for monitoring biotoxins, which are a major concern in both the food safety and the anti-terrorism fields. Current techniques, such as immunochromatographic tests (ICT), enzyme-linked immunosorbent assay (ELISA) and mass spectrometry, are still insufficient to satisfy the needs for fast, label-free, and ultra-sensitive detection. Herein, a single-molecular, label-free detection method based on atomic force microscopy was employed to solve the abovementioned problem via a photo-induced force spectrum; typically, three important biotoxins, i.e. abrin toxin (ABR), ricin toxin (RT) and Clostridium perfringens exotoxin (ETX), were used for the demonstration of single molecule detection. The photo-induced force spectrum could be successfully obtained for each of the single protein particles with molecular weights down to 30 kDa. Furthermore, principal component analysis (PCA) was applied for each protein, resulting in a standard PCA identification database. Then, individual components in a mixture of these toxin proteins were well distinguished from each other via matching with the as-built database. Using this strategy, PiFM not only could be used as a powerful tool for single protein detection, but could also be used as a potential tool in protein structural analysis.