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Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides. Synthesis depends on uridine diphosphate glucose, Mg2+, and cyclic diguanosine monophosphate, with the last displaying a retention time of ~80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.

Biomolecular sensors with single‐molecule resolution are composed of multitudes of transducers that measure state changes related to single‐molecular binding and unbinding events. Conventionally, signals are aggregated from many individual transducers in order to achieve sufficient statistics. However, by aggregating signals, transducer‐to‐transducer differences are lost and heterogeneities cannot be studied. Here, transducers with single‐molecule resolution over long time spans are studied, enabling the collection of sufficient statistics from independent transducers. This allows comparisons between transducers that reveal fundamental heterogeneities in their molecular assemblies related to stochastic variations. The study is performed with biosensing by particle motion, a sensing methodology with thousands of particles that dynamically interact with a sensing surface. The signals of individual particles are studied for series of modulations of analyte concentration over 25 h. The results show large differences in individual concentration‐dependent responses. Monte Carlo simulations clarify that heterogeneities can be attributed to stochastic fluctuations in the numbers of binder molecules, and that gradual changes of the response characteristics can be related to losses of molecules in the single‐particle transducers. The results give insights into molecular and temporal heterogeneities of continuous transducers with single‐molecule resolution and explain how sensors can be engineered to achieve robust, precise, and stable biomolecular monitoring. Methodologies are developed to reveal and understand heterogeneities in continuous biosensors based on a multitude of transducers with single‐molecule resolution. Measurements over long time spans (25 h) show clear inter‐transducer differences, including the shapes of the dose‐response curves. Simulations and time‐dependent changes indicate that the differences are caused by variations in the number of binder molecules per transducer.

DNA–protein interaction plays an essential role in the storage, expression, and regulation of genetic information. A 1D/3D facilitated diffusion mechanism has been proposed to explain the extraordinarily rapid rate of DNA‐binding protein (DBP) searching for cognate sequence along DNA and further studied by single‐molecule experiments. However, direct observation of the detailed chronological protein searching image is still a formidable challenge. Here, for the first time, a single‐molecule electrical monitoring technique is utilized to realize label‐free detection of the DBP–DNA interaction process based on high‐gain silicon nanowire field‐effect transistors (SiNW FETs). The whole binding process of WRKY domain and DNA has been visualized with high sensitivity and single‐base resolution. Impressively, the swinging of hydrogen bonds between amino acid residues and bases in DNA induce the dynamic collective motion of DBP–DNA. This in situ, label‐free electrical detection platform provides a practical experimental methodology for dynamic studies of various biomolecules. A single‐molecule electrical detection platform, based on a silicon nanowire field‐effect transistors (SiNW FETs) nanocircuit, is applied to real‐time monitor the complete interaction process of WRKY1N and DNA with single‐base‐pair resolution. A long‐last collective motion correlated to the specific recognition site is observed at the single‐molecule level, providing a novel technique with single‐molecule/single‐event sensitivity to decipher the comprehensive mechanism of biomolecule interactions.

Little is known about how a non‐Watson–Crick pair affects the RNA folding dynamics. We studied the effects of a U⋅U‐to‐U⋅C pair mutation on the folding of a hairpin in human telomerase RNA. The ensemble thermal melting of the hairpins shows an on‐pathway intermediate with the disruption of the internal loop structure containing the U⋅U/U⋅C pairs. By using optical tweezers, we applied a stretching force on the terminal ends of the hairpins to probe directly the non‐nearest‐neighbour effects upon the mutations. The single U⋅U to U⋅C mutations are observed to 1) lower the mechanical unfolding force by approximately 1 picoNewton (pN) per mutation without affecting the unfolding reaction transition‐state position (thus suggesting that removing a single hydrogen bond affects the structural dynamics at least two base pairs away), 2) result in more frequent misfolding into a small hairpin at approximately 10 pN and 3) shift the folding reaction transition‐state position towards the native hairpin structure and slightly increase the mechanical folding kinetics (thus suggesting that untrapping from the misfolded state is not the rate‐limiting step). In a pinch: The non‐nearest‐neighbour contribution of a single hydrogen bond to RNA folding has been investigated by single‐molecule manipulation using optical tweezers (see figure). Disruption of a single hydrogen bond, by the replacement of a single RNA U⋅U pair with a U⋅C pair, results in an increase in the rate of mechanical unfolding and more frequent misfolding.

Bacterial cells require DNA segregation machinery to properly distribute a genome to both daughter cells upon division. The most common system involved in chromosome and plasmid segregation in bacteria is the ParABS system. A core protein of this system - partition protein B (ParB) - regulates chromosome organization and chromosome segregation during the bacterial cell cycle. Over the past decades, research has greatly advanced our knowledge of the ParABS system. However, many intricate details of the mechanism of ParB proteins were only recently uncovered using in vitro single-molecule techniques. These approaches allowed the exploration of ParB proteins in precisely controlled environments, free from the complexities of the cellular milieu. This review covers the early developments of this field but emphasizes recent advances in our knowledge of the mechanistic understanding of ParB proteins as revealed by in vitro single-molecule methods. Furthermore, we provide an outlook on future endeavors in investigating ParB, ParB-like proteins, and their interaction partners. A review highlighting insights from single-molecule studies on ParB, the key organizing protein that mediates DNA condensation and segregation.

We study the angular fluorescence intensity modulation of a single dye positioned near a spherical gold nanoparticle, induced by rotation of linearly polarized excitation light. Accurate positioning and alignment of nanoparticle and fluorophore with respect to each other and the incoming electric field is achieved by a three-dimensional, self-assembled DNA origami. An intensity map is obtained for a fixed distance and two different nanoparticle diameters, revealing polarization-dependent enhancement and quenching of fluorescence intensity in good agreement to numerical simulations.

The O⁶-alkylguanine DNA alkyltransferase (AGT) is an important DNA repair protein. AGT repairs highly mutagenic and cytotoxic alkylguanine lesions that result from metabolic products but are also deliberately introduced during chemotherapy, making a better understanding of the working mechanism of AGT essential. To investigate lesion interactions by AGT, we present a protocol to insert a single alkylguanine lesion at a well-defined position in long DNA substrates for single-molecule fluorescence microscopy coupled with dual-trap optical tweezers. Our studies address the longstanding enigma in the field of how monomeric AGT complexes at alkyl lesions seen in crystal structures can be reconciled with AGT clusters on DNA at high protein concentrations that have been observed from atomic force microscopy (AFM) and biochemical studies. A role of AGT clusters in enhancing lesion search efficiencies by AGT has previously been proposed. Surprisingly, our data show no enhancement of DNA translocation speed by AGT cluster formation, suggesting that AGT clusters may serve a different role in AGT function. Interestingly, a possible role of these clusters is indicated by preferential cluster formation at alkyl lesions in our studies. From our data, we derive a model for the lesion search and repair mechanism of AGT.

Single‐molecule optoelectronics offers opportunities for advancing integrated photonics and electronics, which also serves as a tool to elucidate the underlying mechanism of light‐matter interaction. Plasmonics, which plays pivotal role in the interaction of photons and matter, have became an emerging area. A comprehensive understanding of the plasmonic excitation and modulation mechanisms within single‐molecule junctions (SMJs) lays the foundation for optoelectronic devices. Consequently, this review primarily concentrates on illuminating the fundamental principles of plasmonics within SMJs, delving into their research methods and modulation factors of plasmon‐exciton. Moreover, we underscore the interaction phenomena within SMJs, including the enhancement of molecular fluorescence by plasmonics, Fano resonance and Rabi splitting caused by the interaction of plasmon‐exciton. Finally, by emphasizing the potential applications of plasmonics within SMJs, such as their roles in optical tweezers, single‐photon sources, super‐resolution imaging, and chemical reactions, we elucidate the future prospects and current challenges in this domain. This study investigates the function of plasmons in single‐molecule junctions. It also analyzes their role in molecular fluorescence enhancement, the interaction between light and matter, and outlines some of their potential applications.

In single molecule study, surface-enhanced Raman scattering (SERS) has the advantage of specifically providing structural information of the molecules targeted. The main challenge in single molecule SERS is developing reusable plasmonic substrates that ensures single molecule sensitivity and acquires intrinsic information of molecules. Here, we proposed a strategy to utilize single-walled carbon nanotubes (SWNTs) to construct SERS substrates. Employing ultrasonic spray pyrolysis, we prepared in situ polyhedral gold nanocrystals closely spaced and attached to nanotubes, ensuring valid hot spots formed along the tube-walls. With such SERS substrates, we proved the single molecule detection by the statistical analysis based on the natural abundance of isotopes. Since SWNTs provide non-chemical bonding adsorption sites, our SERS substrates are easily reusable and have a unique advantage of preserving the intrinsic property of the molecules detected. Using SWNTs to build SERS substrates may become a powerful general strategy in various static and dynamic studies of single molecules.

Magnetism plays a pivotal role in many biological systems. However, the intensity of the magnetic forces exerted between magnetic bodies is usually low, which demands the development of ultra-sensitivity tools for proper sensing. In this framework, magnetic force microscopy (MFM) offers excellent lateral resolution and the possibility of conducting single-molecule studies like other single-probe microscopy (SPM) techniques. This comprehensive review attempts to describe the paramount importance of magnetic forces for biological applications by highlighting MFM’s main advantages but also intrinsic limitations. While the working principles are described in depth, the article also focuses on novel micro- and nanofabrication procedures for MFM tips, which enhance the magnetic response signal of tested biomaterials compared to commercial nanoprobes. This work also depicts some relevant examples where MFM can quantitatively assess the magnetic performance of nanomaterials involved in biological systems, including magnetotactic bacteria, cryptochrome flavoproteins, and magnetic nanoparticles that can interact with animal tissues. Additionally, the most promising perspectives in this field are highlighted to make the reader aware of upcoming challenges when aiming toward quantum technologies.