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The Ebola virus is a deadly pathogen that has been threatening public health for decades. Recent studies have revealed alternative viral invasion routes where Ebola virus approaches cells via interactions among phosphatidylserine (PS), PS binding ligands such as Gas6, and TAM family receptors such as Axl. In this study, we investigate the interactions among phosphatidylserine on the Ebola viral-like particle (VLP) membrane, human Gas6, and human Axl using atomic force microscope-based single molecule force spectroscopy to compare their binding strength and affinity from a biomechanical perspective. The impact of calcium ions on their interactions is also studied and quantified to provide more details on the calcium-dependent phosphatidylserine-Gas6 binding mechanism. Our results indicate that, in the presence of calcium ions, the binding strengths of VLP-Gas6 and VLP-Gas6-Axl increase but are still weaker than that of Gas6-Axl, and the binding affinity of VLP-Gas6 and VLP-Gas6-Axl is largely improved. The binding strength and affinity of Gas6-Axl basically remain the same, indicating no impact in the presence of calcium ions. Together, our study suggests that, under physiological conditions with calcium present, the Ebola virus can utilize its membrane phosphatidylserine to dock on cell surface via Gas6-Axl bound complex. [ABSTRACT FROM AUTHOR]

Cadherins are calcium dependent adhesion proteins that establish and maintain the intercellular mechanical contact by bridging the gap between adjacent cells. Desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) are tissue specific cadherin isoforms of the cell-cell contact in cardiac desmosomes. Mutations in the DSG2 -gene and in the DSC2 -gene are related to arrhythmogenic right ventricular cardiomyopathy (ARVC) a rare but severe heart muscle disease. Here, several possible homophilic and heterophilic binding interactions of wild-type Dsg2, wild-type Dsc2, as well as one Dsg2- and two Dsc2-variants, each associated with ARVC, are investigated. Using single molecule force spectroscopy (SMFS) with atomic force microscopy (AFM) and applying Jarzynski's equality the kinetics and thermodynamics of Dsg2/Dsc2 interaction can be determined. The free energy landscape of Dsg2/Dsc2 dimerization exposes a high activation energy barrier, which is in line with the proposed strand-swapping binding motif. Although the binding motif is not affected by any of the mutations, the binding kinetics of the interactions differ significantly from the wild-type. While wild-type cadherins exhibit an average complex lifetime of approx. 0.3 s interactions involving a variant consistently show - lifetimes that are substantially larger. The lifetimes of the wild-type interactions give rise to the picture of a dynamic adhesion interface consisting of continuously dissociating and (re)associating molecular bonds, while the delayed binding kinetics of interactions involving an ARVC-associated variant might be part of the pathogenesis. Our data provide a comprehensive and consistent thermodynamic and kinetic description of cardiac cadherin binding, allowing detailed insight into the molecular mechanisms of cell adhesion. [Display omitted] • Integrity of heart muscle tissue relies on weak molecular bonds between cadherins. • Single molecule force spectroscopy reveals cadherin binding kinetics. • Wild-type cadherin bonds exhibit characteristic fast (re-) binding kinetics. • Specific cardiomyopathy associated cadherin variants slow binding kinetics. • Impaired molecular dynamics could be a cardiomyopathy-specific pathomechanism. [ABSTRACT FROM AUTHOR]

Single‐molecule force spectroscopy is a powerful tool for the quantitative investigation of the biophysics, polymer physics and mechanochemistry of individual polymer strands. One limitation of this technique is that the attachment between the tip of the atomic force microscope and the covalent or noncovalent analyte in a given pull is typically not strong enough to sustain the force at which the event of interest occurs, which makes the experiments time‐consuming and inhibits throughput. Here we report a polyelectrolyte handle for single‐molecule force spectroscopy that offers a combination of high (several hundred pN) attachment forces, good (~4%) success in obtaining a high‐force (>200 pN) attachment, a non‐fouling detachment process that allows for repetition, and specific attachment locations along the polymer analyte. [ABSTRACT FROM AUTHOR]

BiP (immunoglobulin heavy‐chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide‐binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate‐binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding. Recent structural studies have provided new insights into BiP's allostery; however, the influence of temperature on the coupling between substrate and nucleotide binding to BiP remains unexplored. Here, we study BiP's binding to its substrate at the single molecule level using thermo‐regulated optical tweezers which allows us to mechanically unfold the client protein and explore the effect of temperature and different nucleotides on BiP binding. Our results confirm that the affinity of BiP for its protein substrate relies on nucleotide binding, by mainly regulating the binding kinetics between BiP and its substrate. Interestingly, our findings also showed that the apparent affinity of BiP for its protein substrate in the presence of nucleotides remains invariable over a wide range of temperatures, suggesting that BiP may interact with its client proteins with similar affinities even when the temperature is not optimal. Thus, BiP could play a role as a "thermal buffer" in proteostasis. [ABSTRACT FROM AUTHOR]

Ion-specific effects have a profound impact on the macroscopic properties of polysaccharides such as the aggregation and solubility in solutions. However, the underlying molecular mechanisms still require exploration. In this study, we investigated the influence of anions on the structure and behavior of chitin via a combination of single-molecule techniques and macroscopic characterizations. Our findings reveal that the hydration of anions functions as bridging center, leading to the formation of water-ion-water bridges between chitin side groups. Remarkably, this structure exhibits a binding affinity to chitin following the Hofmeister series with the order of F− < Cl− < Br− < I−, resulting in an escalating of left-handed helix order of chitin. Subsequent experiments indicate that the bridging structure affects the size of chitin aggregates in the similar manner, implying the impact of anions on the structure and behavior of chitin across length scales. These results provide valuable insights into the understanding of ion-specific effects on polysaccharides and its applications in food science where high salt environments are involved. [Display omitted] • Chitin structure and behavior are sensitive to anions. • Chitin interacts with anions via water-ion-water bridges. • Anions prompt the folding of chitin into a left-handed helical structure. • Anions prompt the aggregation of chitin chains. [ABSTRACT FROM AUTHOR]

Simple Summary: Prion diseases are neurodegenerative disorders caused by the amyloidal aggregation of the cellular prion protein. We apply single-molecule force spectroscopy approaches to study the unfolding of prion protein monomers and dimers in different orientations. We find heterogeneous behavior in the prion protein unfolding and an interesting difference between the dimer orientations whereby the dimer in which the C-termini are joined unfolds at a higher force, implying a more stable structure owing to interactions between the C-termini. These results may contribute to a better understanding of the initial steps of oligomer assembly during prion diseases. Prion diseases are neurodegenerative disorders characterized by the presence of oligomers and amyloid fibrils. These are the result of protein aggregation processes of the cellular prion protein (PrPC) into amyloidal forms denoted as prions or PrPSc. We employed atomic force microscopy (AFM) for single molecule pulling (single molecule force spectroscopy, SMFS) experiments on the recombinant truncated murine prion protein (PrP) domain to characterize its conformations and potential initial oligomerization processes. Our AFM-SMFS results point to a complex scenario of structural heterogeneity of PrP at the monomeric and dimer level, like other amyloid proteins involved in similar pathologies. By applying this technique, we revealed that the PrP C-terminal domain unfolds in a two-state process. We used two dimeric constructs with different PrP reciprocal orientations: one construct with two sequential PrP in the N- to C-terminal orientation (N-C dimer) and a second one in the C- to C-terminal orientation (C-C dimer). The analysis revealed that the different behavior in terms of unfolding force, whereby the dimer placed C-C dimer unfolds at a higher force compared to the N-C orientation. We propose that the C-C dimer orientation may represent a building block of amyloid fibril formation. [ABSTRACT FROM AUTHOR]

Hydrogels are crosslinked polymer networks with time-dependent mechanical response. The overall mechanical properties are correlated with the dynamics of the crosslinks. Generally, hydrogels crosslinked by permanent chemical crosslinks are strong but static, while hydrogels crosslinked by physical interactions are weak but dynamic. It is highly desirable to create synthetic hydrogels that possess strong mechanical stability yet remain dynamic for various applications, such as drug delivery cargos, tissue engineering scaffolds, and shape-memory materials. Recently, with the introduction of dynamic covalent chemistry, the seemingly conflicting mechanical properties, i.e., stability and dynamics, have been successfully combined in the same hydrogels. Dynamic covalent bonds are mechanically stable yet still capable of exchanging, dissociating, or switching in response to external stimuli, empowering the hydrogels with self-healing properties, injectability and suitability for postprocessing and additive manufacturing. Here in this review, we first summarize the common dynamic covalent bonds used in hydrogel networks based on various chemical reaction mechanisms and the mechanical strength of these bonds at the single molecule level. Next, we discuss how dynamic covalent chemistry makes hydrogel materials more dynamic from the materials perspective. Furthermore, we highlight the challenges and future perspectives of dynamic covalent hydrogels. [ABSTRACT FROM AUTHOR]

We performed single molecule force spectroscopy assays to elucidate the competition between the two binding modes (intercalation and groove binding) that occur between the drug chloroquine (CLQ) and DNA in HEPES-based buffers. Force–extension curves were obtained under different drug concentrations and buffer compositions, allowing the determination of the changes on the mechanical parameters of these complexes and the physical chemistry of the interactions. The presence of HEPES in the buffer, when combined with NaCl, can modulate the interaction. The conclusions allowed us to advance in the understanding of the role of the driving forces in the balance between distinct binding modes. [Display omitted] • Chloroquine is a complex DNA ligand that can interact by different binding modes. • The effective binding can be modulated by changing the buffer ionic strength and composition. • We advance in the understanding of the specific role of the driving forces in the balance between distinct binding modes. [ABSTRACT FROM AUTHOR]

We performed a robust characterization of the molecular interactions between the DNA molecule and two imidazolium-based ionic liquids (ILs): 1-Butyl-3-methylimidazolium chloride ([bmim]Cl) and 1-Octyl-3-methylimidazolium chloride ([omim]Cl), using single molecule approaches (optical and magnetic tweezers) and bulk techniques (isothermal titration calorimetry and conductivity measurements). Optical and magnetic tweezers allowed us to obtain the changes on the mechanical properties of the DNA complexes formed with both ILs, as well as the relevant physicochemical (binding) parameters of the interaction. Despite the weak binding measured between DNA and the two ILs, we identify a transition on the regime of polymer elasticity of the complexes formed, which results in a relevant DNA compaction for high IL concentrations. In addition, isothermal titration calorimetry and conductivity complemented the single molecule investigation, giving a complete thermodynamic characterization of the interactions and allowing the identification of the most relevant driving forces at various different concentration ranges of the ILs. Based on the results obtained with all the employed techniques, we propose a model for the binding schemes involving DNA and both [bmim]Cl and [omim]Cl. [ABSTRACT FROM AUTHOR]

Abstract Background Sialyl-Lewis X/L-selectin high affinity binding interactions between transmembrane O-glycosylated mucins proteins and the embryo have been implicated in implantation processes within the human reproductive system. However, the adhesive properties of these mucins at the endometrial cell surface are difficult to resolve due to known discrepancies between in vivo models and the human reproductive system and a lack of sensitivity in current in vitro models. To overcome these limitations, an in vitro model of the human endometrial epithelial was interrogated with single molecule force spectroscopy (SMFS) to delineate the molecular configurations of mucin proteins that mediate the high affinity L-selectin binding required for human embryo implantation. Results This study reveals that MUC1 contributes to both the intrinsic and extrinsic adhesive properties of the HEC-1 cellular surface. High expression of MUC1 on the cell surface led to a significantly increased intrinsic adhesion force (148 pN vs. 271 pN, p 400 pN) L-selectin binding at the cell surface, low expression of MUC1 with reduced glycosylation resulted in significantly less (≤200 pN) binding events. Conclusions An optimal level of MUC1 together with highly glycosylated decoration of the protein is critical for high affinity L-selectin binding. This study demonstrates that MUC1 contributes to cellular adhesive properties which may function to facilitate trophoblast binding to the endometrial cell surface through the L-selectin/sialyl-Lewis x adhesion system subsequent to implantation.

In this work, the single-chain elasticity of polyformaldehyde (POM) is studied, for the first time, by employing atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS). We find that the single-chain elasticity of POM in a nonpolar organic solvent (nonane) can be described well by a theoretical model (QM-FRC model), when the rotating unit length is 0.144 nm (C-O bond length). After comparison, POM is more flexible than polystyrene (a typical polymer with C-C backbone) at the single-chain level, which is reasonable since the C-O bond has a lower rotation barrier than C-C bond. This result indicates that the flexibility of a polymer chain can be tuned by the C-O bond proportion in backbone, which casts new light on the rational design of new synthetic polymers in the future. [ABSTRACT FROM AUTHOR]

• AFM stretching and SMD simulations are combined to study protein folding pathways. • Coiled-coils probes for the identification of the unfolding pathways of proteins are described. • Theoretical background and sample preparation for proteins force spectroscopy are provided. Most proteins in proteomes are large, typically consist of more than one domain and are structurally complex. This often makes studying their mechanical unfolding pathways challenging. Proteins composed of tandem repeat domains are a subgroup of multi-domain proteins that, when stretched, display a saw-tooth pattern in their mechanical unfolding force extension profiles due to their repetitive structure. However, the assignment of force peaks to specific repeats undergoing mechanical unraveling is complicated because all repeats are similar and they interact with their neighbors and form a contiguous tertiary structure. Here, we describe in detail a combination of experimental and computational single-molecule force spectroscopy methods that proved useful for examining the mechanical unfolding and refolding pathways of ankyrin repeat proteins. Specifically, we explain and delineate the use of atomic force microscope-based single molecule force spectroscopy (SMFS) to record the mechanical unfolding behavior of ankyrin repeat proteins and capture their unusually strong refolding propensity that is responsible for generating impressive refolding force peaks. We also describe Coarse Grain Steered Molecular Dynamic (CG-SMD) simulations which complement the experimental observations and provide insights in understanding the unfolding and refolding of these proteins. In addition, we advocate the use of novel coiled-coils-based mechanical polypeptide probes which we developed to demonstrate the vectorial character of folding and refolding of these repeat proteins. The combination of AFM-based SMFS on native and CC-equipped proteins with CG-SMD simulations is powerful not only for ankyrin repeat polypeptides, but also for other repeat proteins and more generally to various multidomain, non-repetitive proteins with complex topologies. [ABSTRACT FROM AUTHOR]

Hydrogels are crosslinked polymer networks with time-dependent mechanical response. The overall mechanical properties are correlated with the dynamics of the crosslinks. Generally, hydrogels crosslinked by permanent chemical crosslinks are strong but static, while hydrogels crosslinked by physical interactions are weak but dynamic. It is highly desirable to create synthetic hydrogels that possess strong mechanical stability yet remain dynamic for various applications, such as drug delivery cargos, tissue engineering scaffolds, and shape-memory materials. Recently, with the introduction of dynamic covalent chemistry, the seemingly conflicting mechanical properties, i.e., stability and dynamics, have been successfully combined in the same hydrogels. Dynamic covalent bonds are mechanically stable yet still capable of exchanging, dissociating, or switching in response to external stimuli, empowering the hydrogels with self-healing properties, injectability and suitability for postprocessing and additive manufacturing. Here in this review, we first summarize the common dynamic covalent bonds used in hydrogel networks based on various chemical reaction mechanisms and the mechanical strength of these bonds at the single molecule level. Next, we discuss how dynamic covalent chemistry makes hydrogel materials more dynamic from the materials perspective. Furthermore, we highlight the challenges and future perspectives of dynamic covalent hydrogels.

Titin is a giant elastic protein which is responsible for passive muscle stiffness when muscle sarcomeres are stretched. Chloramphenicol, besides being a broad-spectrum antibiotic, also acts as a muscle relaxant. Therefore, it is important to study the interaction between titin I27 and chloramphenicol. We investigated the interaction of chloramphenicol with octamer of titin I27 using single-molecule force spectroscopy and fluorescence spectroscopy. The fluorescence data indicated that binding of chloramphenicol with I27 results in fluorescence quenching. Furthermore, it is observed that chloramphenicol binds to I27 at a particular concentration (∼ 40 μM). Single-molecule force spectroscopy shows that, in the presence of 40 μM chloramphenicol concentration, the I27 monomers become mechanically stable, resulting in an increment of the unfolding force. The stability was further confirmed by chemical denaturation experiments on monomers of I27, which corroborate the evidence for enhanced mechanical stability at 40 μM drug concentration. The free energy of stabilization for I27 (wild type) was found to be 1.95 ± 0.93 kcal/mole and I27 with 40 μM drug was 3.25 ± 0.63 kcal/mole. The results show a direct effect of the broad-spectrum antibiotic chloramphenicol on the passive elasticity of muscle protein titin. The I27 is stabilized both mechanically and chemically by chloramphenicol. [ABSTRACT FROM AUTHOR]

Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer's disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu(2+). Cu(2+) cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu(2+) regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu(2+) cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu(2+) cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu(2+) cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu(2+) cations induce Aβ42 peptide structural changes resulting in N-termini interactions within the dimers. Cu(2+) cations also stabilize the dimers. No effects of Cu(2+) cations on Aβ-Aβ interactions were observed at pH 4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu(2+) cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu(2+) cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu(2+) cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu(2+) cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu(2+).

Immunoassays typically must be stored under refrigerated conditions because antibodies, after being immobilized to solid surfaces, tend to lose their recognition capabilities to target antigens under non-refrigerated conditions. This requirement hinders application of immunoassays in resource-limited settings including rural clinics in tropical regions, disaster struck areas, and low-income countries, where refrigeration may not be feasible. In this work, a facile approach based on a reversable zeolitic imidazolate framework-8 (ZIF-8) coating is introduced to stabilize surface-bound antibodies on enzyme-linked immunosorbent assay (ELISA) plates under non-refrigerated conditions. Using a sandwich ELISA for the detection of neutrophil gelatinase-associated lipocalin (NGAL), a urine biomarker for acute kidney injury, as a model system, ZIF-8 is demonstrated to be able to uniformly coat the surface-bound anti-NGAL IgG, and stabilize the dynamic range and detection sensitivity of the assay after storage at an elevated temperature (50 °C) for at least 4 weeks. The stabilization efficacy of the ZIF-8 coating is comparable to the current "gold standard" refrigeration approach, and superior to the commonly used sucrose coating method. This approach will greatly improve the shelf-life and stability of antibody-coated ELISAs and other types of assays which utilize surface-bound antibodies, thus extending biomedical research and medical diagnostics to resource-limited settings. [ABSTRACT FROM AUTHOR]

Crystal structures and experiments relying on the tools of molecular pharmacology reported conflicting results on ligand binding sites in neurotransmitter/sodium symporters (NSS). We explored the number and functionality of ligand binding sites of NSS in a physiological setting by designing novel tools for atomic force microscopy (AFM). These allow for directly measuring the interaction forces between the serotonin transporter (SERT) and the antidepressant S-citalopram (S-CIT) on the single molecule level: the AFM cantilever tips were functionalized with S-CIT via a flexible polyethylene glycol (PEG) linker. The tip chemistry was validated by specific force measurements and recognition imaging on CHO cells. Two distinct populations of characteristic binding strengths of S-CIT binding to SERT were revealed in Na+-containing buffer. In contrast, in Li+-containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT. The dissociation rate constant of both binding sites was extracted by varying the dynamics of the force-probing experiments. Competition experiments revealed that the two sites are allosterically coupled and exert reciprocal modulation.

Background: Sialyl-Lewis X/L-selectin high affinity binding interactions between transmembrane O-glycosylated mucins proteins and the embryo have been implicated in implantation processes within the human reproductive system. However, the adhesive properties of these mucins at the endometrial cell surface are difficult to resolve due to known discrepancies between in vivo models and the human reproductive system and a lack of sensitivity in current in vitro models. To overcome these limitations, an in vitro model of the human endometrial epithelial was interrogated with single molecule force spectroscopy (SMFS) to delineate the molecular configurations of mucin proteins that mediate the high affinity L-selectin binding required for human embryo implantation. Results: This study reveals that MUC1 contributes to both the intrinsic and extrinsic adhesive properties of the HEC-1 cellular surface. High expression of MUC1 on the cell surface led to a significantly increased intrinsic adhesion force (148 pN vs. 271 pN, p < 0.001), whereas this adhesion force was significantly reduced (271 pN vs. 118 pN, p < 0.001) following siRNA mediated MUC1 ablation. Whilst high expression of MUC1 displaying elevated glycosylation led to strong extrinsic (> 400 pN) L-selectin binding at the cell surface, low expression of MUC1 with reduced glycosylation resulted in significantly less (≤200 pN) binding events. Conclusions: An optimal level of MUC1 together with highly glycosylated decoration of the protein is critical for high affinity L-selectin binding. This study demonstrates that MUC1 contributes to cellular adhesive properties which may function to facilitate trophoblast binding to the endometrial cell surface through the L-selectin/sialyl-Lewis x adhesion system subsequent to implantation. [ABSTRACT FROM AUTHOR]

Supramolecular helices have unique properties and many potential applications, such as chiral separation and asymmetric catalysis. Mechanical property (stability) of the supramolecular helix plays important roles in their functions. Due to the limitation of detection method, it is quite challenging to investigate nanomechanical properties of individual supramolecular helices stabilized by pure supramolecular interactions. Here atomic force microscopy (AFM)‐based single molecule force spectroscopy (SMFS) is used to study the nanomechanical properties of a thermal‐responsive supramolecular helix. The unwinding force plateau is observed in the force‐extension curve, and the rupture force of the helix is dependent on the loading rate. In addition, the force‐induced unwinding process is reversible and there is almost no energy dissipation in the process. Furthermore, the result of thermal shape‐fluctuation analysis shows that the persistence length of the supramolecular helix is about 222 nm, which is much larger than helical structure formed by double‐stranded DNA (dsDNA). However, because of its unique backbone structure, the supramolecular helix exhibits higher dynamic flexibility during force‐induced deformation, since the persistence length determined from the stretching experiment is much smaller (1.1 nm). [ABSTRACT FROM AUTHOR]

Europium oxide (Eu 2 O 3) was used to evaluate the affinity of this rare earth element for interacting with double-stranded (ds) DNA molecules. To perform the study, we used single molecule force spectroscopy with optical tweezers and gel electrophoresis assays. Force spectroscopy experiments show that Eu 2 O 3 presents a strong interaction with dsDNA, and the binding is independent on the ionic strength used in the surrounding environment. Among the main characteristics of the interaction, Eu 2 O 3 tends to bind in a cooperative way, forming bound clusters of ∼ 3 molecules, and presents a high equilibrium association binding constant on the order of 105 M−1. In addition, gel electrophoresis confirm the weak electrostatic character of the interaction and explicit show that Eu 2 O 3 does not interfere on drug intercalation into the double-helix. Such results demonstrate the potential of europium for interacting with nucleic acids and strongly suggest that this rare earth element may be considered for the design of new metal-based anticancer drugs in the future. Image 1 • Europium oxide binds strongly and cooperatively to double-stranded DNA. • The binding mode and the physical chemistry of the interaction were depicted using single molecule force spectroscopy. • The study suggest that rare earth elements are promising to the rational design of new metal-based anticancer drugs. [ABSTRACT FROM AUTHOR]

Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF165 was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF165 was dependent on the concentration of VEGF165. On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF165 complexes was revealed, which was similar to that of the intact aptamer/VEGF165. Besides, the dissociation rate constant (koff) of split aptamer/VEGF165 was close to that of intact aptamer/VEGF165, and the interaction force of split aptamer/VEGF165 was higher than the force of intact aptamer/VEGF165. It indicated that split aptamer also possessed high affinity with VEGF165. The work can provide a new method for exploring the interaction of split aptamer/its targets at single‐molecule level. [ABSTRACT FROM AUTHOR]

Abstract Background In the past two decades, methods have been developed to measure the mechanical properties of single biomolecules. One of these methods, Magnetic tweezers, is amenable to acquisition of data on many single molecules simultaneously, but to take full advantage of this "multiplexing" ability, it is necessary to simultaneously incorporate many capabilities that have been only demonstrated separately. Methods Our custom built magnetic tweezer combines high multiplexing, precision bead tracking, and bi-directional force control into a flexible and stable platform for examining single molecule behavior. This was accomplished using electromagnets, which provide high temporal control of force while achieving force levels similar to permanent magnets via large paramagnetic beads. Results Here we describe the instrument and its ability to apply 2–260 pN of force on up to 120 beads simultaneously, with a maximum spatial precision of 12 nm using a variety of bead sizes and experimental techniques. We also demonstrate a novel method for increasing the precision of force estimations on heterogeneous paramagnetic beads using a combination of density separation and bi-directional force correlation which reduces the coefficient of variation of force from 27% to 6%. We then use the instrument to examine the force dependence of uncoiling and recoiling velocity of type 1 fimbriae from Eschericia coli (E. coli) bacteria, and see similar results to previous studies. Conclusion This platform provides a simple, effective, and flexible method for efficiently gathering single molecule force spectroscopy measurements.

Here, we use single molecule force spectroscopy performed with optical tweezers in order to investigate the interaction between Caffeine and the DNA molecule for various different concentrations of the alkaloid and under two distinct ionic strengths of the surrounding buffer. We were able to determine the mechanical changes induced on the double-helix structure due to Caffeine binding, the binding mode and the binding parameters of the interaction. The results obtained show that Caffeine binds to DNA by outside the double-helix with a higher affinity at lower ionic strengths. On the other hand, a considerable cooperativity was found only for sufficient high ionic strengths, suggesting that Caffeine may binding forming dimers and/or trimers along the double-helix under this condition. Finally, it was also shown that Caffeine stabilizes the DNA double-helix upon binding, preventing force-induced DNA melting. [ABSTRACT FROM AUTHOR]

Summary: Real‐time tracking of multiple particles is key for quantitative analysis of dynamic biophysical processes and materials science via time‐lapse microscopy image data, especially for single molecule biophysical techniques, such as magnetic tweezers and centrifugal force microscopy. However, real‐time multiple particle tracking with high resolution is limited by the current imaging processes or tracking algorithms. Here, we demonstrate 1 nm resolution in three dimensions in real‐time with a graphics‐processing unit (GPU) based on a compute unified device architecture (CUDA) parallel computing framework instead of only a central processing unit (CPU). We also explore the trade‐offs between processing speed and size of the utilized regions of interest and a maximum speedup of 137 is achieved with the GPU compared with the CPU. Moreover, we utilize this method with our recently self‐built centrifugal force microscope (CFM) in experiments that track multiple DNA‐tethered particles. Our approach paves the way for high‐throughput single molecule techniques with high resolution and efficiency. Lay Description: Particles are widely used as probes in life sciences through their motions. In single molecule techniques such as optical tweezers and magnetic tweezers, microbeads are used to study intermolecular or intramolecular interactions via beads tracking. Also tracking multiple beads' motions could study cell–cell or cell–ECM interactions in traction force microscopy. Therefore, particle tracking is of key important during these researches. However, parallel 3D multiple particle tracking in real‐time with high resolution is a challenge either due to the algorithm or the program. Here, we combine the performance of CPU and CUDA‐based GPU to make a hybrid implementation for particle tracking. In this way, a speedup of 137 is obtained compared the program before only with CPU without loss of accuracy. Moreover, we improve and build a new centrifugal force microscope for multiple single molecule force spectroscopy research in parallel. Then we employed our program into centrifugal force microscope for DNA stretching study. Our results not only demonstrate the application of this program in single molecule techniques, also indicate the capability of multiple single molecule study with centrifugal force microscopy. [ABSTRACT FROM AUTHOR]

We perform a single molecule force spectroscopy study to characterize the role of the size (molecular weight) and concentration of depleting agents on DNA condensation and on the stabilization of the double-helix structure, showing that important features such as the threshold concentration for DNA condensation, the force in which the melting plateau occurs and its average length strongly depend on the depletant size chosen. Such results are potentially important to understand how the presence of surrounding macromolecules influences DNA stabilization inside living cells and therefore advance in the understanding of the crowded cell environment on DNA-related functions. [Display omitted] • Depleting agents play a role on the stabilization of the double-helix structure. • Force-induced melting strongly depends on the depletant size and concentration. • Results advance in the understanding of DNA stabilization inside cells. [ABSTRACT FROM AUTHOR]

Here we report a new study performed at single molecule level on the interaction of the antineoplastic drug Carboplatin and the DNA molecule - the main target of the drug inside cells in cancer chemotherapies. By using optical tweezers, we measure how the mechanical properties of the DNA-Carboplatin complexes changes as a function of the drug concentration in the sample, for two different ionic strengths ([Na] = 150 mM and [Na] = 1 mM). From these measurements, the binding mechanism and the physicochemical (binding) parameters of the interaction were inferred and directly compared to those obtained for the precursor drug Cisplatin under equivalent conditions. As the main conclusion, we show that Carboplatin binds preferentially forming covalent monoadducts in contrast to Cisplatin, which is hydrolyzed easier and presents a higher efficiency in forming covalent diadducts along the double-helix. In addition, we explicitly show that Carboplatin is much less sensitive to ionic strength changes when compared to Cisplatin. These findings provide new insights on the interactions of platinum-based compounds with the DNA molecule, being important to improve the current treatments and in the development of new antineoplastic agents. [ABSTRACT FROM AUTHOR]

Abstract: Protein aggregation is linked with the onset of several neurodegenerative disorders, including Parkinson's disease (PD), which is associated with the aggregation of α‐synuclein (αSyn). The structural mechanistic details of protein aggregation, including the nature of the earliest protein–protein interactions, remain elusive. In this study, we have used single molecule force spectroscopy (SMFS) to probe the first dimerization events of the central aggregation‐prone region of αSyn (residues 71–82) that may initiate aggregation. This region has been shown to be necessary for the aggregation of full length αSyn and is capable of forming amyloid fibrils in isolation. We demonstrate that the interaction of αSyn71‐82 peptides can be studied using SMFS when inserted into a loop of protein L, a mechanically strong and soluble scaffold protein that acts as a display system for SMFS studies. The corresponding fragment of the homolog protein γ‐synuclein (γSyn), which has a lower aggregation propensity, has also been studied here. The results from SMFS, together with native mass spectrometry and aggregation assays, demonstrate that the dimerization propensity of γSyn71‐82 is lower than that of αSyn71‐82, but that a mixed αSyn71‐82: γSyn71‐82 dimer forms with a similar propensity to the αSyn71‐82 homodimer, slowing amyloid formation. This work demonstrates the utility of a novel display method for SMFS studies of aggregation‐prone peptides, which would otherwise be difficult to study. [ABSTRACT FROM AUTHOR]

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We have performed a biophysical characterization, at single molecule level, of the interaction between the DNA molecule and the biogenic polyamine putrescine. By using force spectroscopy, we were able to monitor the complexes formation as putrescine is added to the sample, determining the mechanical properties of such complexes and the physicochemical (binding) parameters of the interaction for three different ionic strengths. In particular, it was shown that the behavior of the equilibrium binding constant as a function of the counterion concentration deviates from the prediction of the Record-Lohman model. The measured constants were (1.3 ± 0.2) × 10 5  M - 1 for [Na] = 150 mM, (2.1 ± 0.2) × 10 5  M - 1 for [Na] = 10 mM, and (2.2 ± 0.3) × 10 5  M - 1 for [Na] = 1 mM. The cooperativity degree of the binding reaction, on the other hand, increases with the ionic strength. From these analysis, the DNA-putrescine binding mechanisms are inferred, and a comparison with results reported for ordinary bivalent ions like magnesium is performed. Such study provides new insights on the general behavior of the DNA interactions with biogenic polyamines. [ABSTRACT FROM AUTHOR]

Many biological processes rely on protein–membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach, single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2–7 pN and had binding energies of 4–14 kBT per C2 domain. Regulation by bilayer composition or Ca2+ recapitulated known properties of both proteins. The method can be widely applied to study protein–membrane interactions.

The present article offers an overview on the use of atomic force microscopy (AFM) to characterize the nanomechanical properties of polymers. AFM imaging reveals the conformations of polymer molecules at solid– liquid interfaces. In particular, for polyelectrolytes, the effect of ionic strength on the conformations of molecules can be studied. Examination of force versus extension profiles obtained using AFM-based single molecule force spectroscopy gives information on the entropic and enthalpic elasticities in pN to nN force range. In addition, single molecule force spectroscopy can be used to trigger chemical reactions and transitions at the molecular level when force-sensitive chemical units are embedded in a polymer backbone.

From the first experiments of the atomic force microscopy (AFM) with biological samples, the range of its potential applications grows extensively. One of them is the use of AFM to characterize biophysical fingerprints of cancer progression in search of non-labelled biomarkers of the disease. The technique offers various functionalities, starting from surface imaging to detection of interaction forces, delivering quantitative parameters that can describe changes characteristic for various diseases, including cancer. In this review, the special emphasis was laid on these studies that compare the AFM-derived properties of reference and cancerous cells using all functionalities from cellular deformability measurements to quantification of the interaction forces at the single-molecule and single-cell levels. Despite the large effort and evidence of the microscope applicability to detect pathologically altered cells, there are still practical challenges remained to be solved before AFM can be implemented for routine cancer tracking and diagnosis. To-date, the AFM can be used to achieve a better understanding of cancer-related processes and mechanisms that could be further employed to design high-resolution clinical assays in a quantitative way. [ABSTRACT FROM AUTHOR]

The cell membrane, involved in almost all communications of cells and surrounding matrix, is one of the most complicated components of cells. Lack of suitable methods for the detection of cell membranes in vivo has sparked debates on the biochemical composition and structure of cell membranes over half a century. The development of single molecule techniques, such as AFM, SMFS, and TREC, provides a versatile platform for imaging and manipulating cell membranes in biological relevant environments. Here, we discuss the latest developments in AFM and the progress made in cell membrane research. In particular, we highlight novel structure models and dynamic processes, including the mechanical properties of the cell membranes. [ABSTRACT FROM AUTHOR]

It is now possible to manipulate individual molecules using a nanopore to read DNA and proteins, or write DNA by inserting mini-genes into cells. Furthermore, development of these methodologies will kick open the door to new biology and chemistry that has been logistically intractable previously. Nanopore technology will place molecular and sub-molecular analysis within the reach of the typical bench-top scientist or clinical lab-no longer limited to genomics or mass spectrometry specialists. Moreover, the prospects for synthetic biology-using nanopores to program or reprogram cells-are promising as well, but have been examined only at the level of a single cell, so far.