Supramolecular mechanophores typically exhibit much lower mechanical strengths than covalent counterparts, with strengths usually around 100 pN, which is significantly lower than the nN-scale strength of covalent bonds. Inspired by the slow dissociation kinetics of the cucurbit[7]uril (CB[7])-hexanoate-isoquinoline (HIQ) complex, we discovered that charge-dipole repulsion can be utilized to create strong supramolecular mechanophores. When activated at its -COO- state, the CB[7]-HIQ complex exhibits a high mechanical strength of ~700 pN, comparable to weak covalent bonds such as Au-S bonds or thiol-maleimide adducts. The strength of the CB[7]-HIQ complex can also be tuned with pH in a gradual manner, with a minimum value of ~150 pN at its -COOH state, similar to an ordinary supramolecular conjugate. This research may pave the way for the development of supramolecular architectures that combine the advantages of covalent and supramolecular systems.

Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe\'s robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.

Recent research on mechano-radicals has provided valuable insights into self-growth and adaptive responsive materials. Typically, mechanophores must remain inert in the absence of force but respond quickly to external tension before other linkages within the polymer network. Azo compounds exhibit promising combinations of mechanical stability and force-triggered reactivity, making them widely used as mechano-radicals in force-responsive materials. However, the activation conditions and behavior of azo compounds have yet to be quantitatively explored. In this study, we investigated the mechanical strength of three azo compounds using single-molecule force spectroscopy. Our results revealed that these compounds exhibit rupture forces ranging from ~500 to 1000 pN, at a loading rate of 3×104 pN s-1. Importantly, these mechanophores demonstrate distinct kinetic properties. Their unique mechanical attributes enable azo bond scission and free radical generation before causing major polymer backbone damage of entire material during polymer network deformation. This fundamental understanding of mechanophores holds significant promise for the development of self-growth materials and their related applications.

Typical methods for stable immobilization of proteins often involve time-consuming surface modification of silicon-based materials to enable specific binding, while the nonspecific adsorption method is faster but usually unstable. Herein, we fused a silica-binding protein, Si-tag, to target proteins so that the target proteins could attach directly to silica substrates in a single step, markedly streamlining the immobilization process. The adhesion force between the Si-tag and glass substrates was determined to be approximately 400-600 pN at the single-molecule level by atomic force microscopy, which is greater than the unfolding force of most proteins. The adhesion force of the Si-tag exhibits a slight increase when pulled from the C-terminus compared to that from the N-terminus. Furthermore, the Si-tag\'s adhesion force on a glass surface is marginally higher than that on a silicon nitride probe. The binding properties of the Si-tag are not obviously affected by environmental factors, including pH, salt concentration, and temperature. In addition, the macroscopic adhesion force between the Si-tag-coated hydrogel and glass substrates was ∼40 times higher than that of unmodified hydrogels. Therefore, the Si-tag, with its strong silica substrate binding ability, provides a useful tool as an excellent fusion tag for the rapid and mechanically robust immobilization of proteins on silica and for the surface coating of silica-binding materials.

CLEC12A, a member of the C-type lectin receptor family involved in immune homeostasis, recognizes MSU crystals released from dying cells. However, the molecular mechanism underlying the CLEC12A-mediated recognition of MSU crystals remains unclear. Herein, we reported the crystal structure of the human CLEC12A-C-type lectin-like domain (CTLD) and identified a unique \"basic patch\" site on CLEC12A-CTLD that is necessary for the binding of MSU crystals. Meanwhile, we determined the interaction strength between CLEC12A-CTLD and MSU crystals using single-molecule force spectroscopy. Furthermore, we found that CLEC12A clusters at the cell membrane and seems to serve as an internalizing receptor of MSU crystals. Altogether, these findings provide mechanistic insights for understanding the molecular mechanisms underlying the interplay between CLEC12A and MSU crystals.

Polyethylene glycol (PEG) is an artificial polymer with good biocompatibility and a low cost, which has a wide range of applications. In this study, the dynamic response of PEG single chains to different ion concentrations was investigated from a microscopic point of view based on single-molecule force spectroscopy, revealing unique interactions that go beyond the traditional sensor-design paradigm. Under low concentrations of potassium chloride, PEG single chains exhibit a gradual reduction in rigidity, while, conversely, high concentrations induce a progressive increase in rigidity. This dichotomy serves as the cornerstone for a profound understanding of PEG conformational dynamics under diverse ion environments. Capitalizing on the remarkable sensitivity of PEG single chains to ion concentration shifts, we introduce innovative sensor-design ideas. Rooted in the adaptive nature of PEG single chains, these sensor designs extend beyond the traditional applications, promising advancements in environmental monitoring, healthcare, and materials science.

Chitin is one of the most common polysaccharides and is abundant in the cell walls of fungi and the shells of insects and aquatic organisms as a skeleton. The mechanism of how chitin responds to pH is essential to the precise control of brewing and the design of smart chitin materials. However, this molecular mechanism remains a mystery. Results from single-molecule studies, including single-molecule force spectroscopy (SMFS), AFM imaging, and molecular dynamic (MD) simulations, have shown that the mechanical and conformational behaviors of chitin molecules show surprising pH responsiveness. This can be compared with how, in natural aqueous solutions, chitin tends to form a more relaxed spreading conformation and show considerable elasticity under low stretching forces in acidic conditions. However, its molecular chain collapses into a rigid globule in alkaline solutions. The results show that the chain state of chitin can be regulated by the proportions of inter- and intramolecular H-bonds, which are determined via the number of water bridges on the chain under different pH values. This basic study may be helpful for understanding the cellular activities of fungi under pH stress and the design of chitin-based drug carriers.

Due to the dynamic nature of ester linkages, ester-bond-containing materials are well known for their outstanding degradability and stimuli responsiveness. However, whether ester hydrolysis is affected by mechanical forces remains unclear. Here, we develop a single-molecule assay to quantitatively study the force-dependent ester hydrolysis using an engineered circular permutant protein with a caged ester bond as a model. Our single-molecule force spectroscopy results show that the ester hydrolysis rate is surprisingly insensitive to forces, with a ∼7 s-1 dissociation rate that remains almost unchanged in the force range of 80 to 200 pN. Quantum calculations reveal that the ester hydrolysis involves an intermediate state formed by either H3O+- or OH--bonded tetrahedral intermediates. The measured ester-hydrolysis kinetics at the single-molecule level may primarily correspond to the rupture of these intermediate states. However, the rate-limiting step appears to be the formation of the tetrahedral intermediates, which cannot be quantitatively characterized in our experiments. Nonetheless, based on the quantum calculations, this step is also insensitive to applied forces. Altogether, our study suggests that the ester bond is chemically labile yet mechanically stable, serving as the basis for the design of responsive materials using ester bonds as mechanically inert units.

Nonspecific interactions play a significant role in physiological activities, surface chemical modification, and artificial adhesives. However, nonspecificity sometimes causes sticky problems, including surface fouling, decreased target specificity, and artifacts in single-molecule measurements. Adjusting the liquid pH, using protein-blocking additives, adding nonionic surfactants, or increasing the salt concentration are common methods to minimize nonspecific binding to achieve high-quality data. Here, we report that grafting heteromorphic polyethylene glycol (Y-shape PEG) with two inert terminates could noticeably decrease nonspecific binding. As a proof-of-concept, we performed single-molecule force spectroscopy and fluorescence staining imaging experiments to verify the feasibility of Y-shape PEG in blocking nonspecific interactions. Our results indicate that Y-shape PEG could serve as a prominent and efficient candidate to minimize nonspecificity for scientific and biomedical applications.

Ribonucleic acid (RNA) and lipid are essential biomolecules in many biological processes, and hold a great prospect for biomedical applications, such as gene therapy, vaccines and therapeutic drug delivery. The characterization of morphology and intra-/inter-molecular interactions of RNA and lipid molecules is critical for understanding their functioning mechanisms. Atomic force microscopy (AFM) is a sophisticated technique for characterizing biomolecules featured by its piconewton force sensitivity, sub-nanometer spatial resolution, and flexible operation conditions in both air and liquid. The goal of this review is to highlight the representative and outstanding discoveries of the characterization of RNA and lipid molecules through morphology identification, physicochemical property determination and intermolecular force measurements by AFM. The first section introduces the AFM imaging of RNA molecules to obtain high-resolution morphologies and nanostructures in air and liquid, followed by the discussion of employing AFM force spectroscopy in understanding the nanomechanical properties and intra-/inter-molecular interactions of RNA molecules, including RNA-RNA and RNA-biomolecule interactions. The second section focuses on the studies of lipid and RNA encapsulated in lipid carrier (RNA-lipid) by AFM as well as the sample preparation and factors influencing the morphology and structure of lipid/RNA-lipid complexes. Particularly, the nanomechanical properties of lipid and RNA-lipid characterized by nanomechanical imaging and force measurements are discussed. The future perspectives and remaining challenges on the characterization of RNA and lipid offered by the versatile AFM techniques are also discussed. This review provides useful insights on the characterization of RNA and lipids nanostructures along with their molecular interactions, and also enlightens the application of AFM techniques in investigating a broad variety of biomolecules.