Mechanophores are powerful molecular tools used to track bond rupture and characterize mechanical damage in polymers. The majority of mechanophores are known to respond to external stresses, and we report in this study the first precedent of a mechanochemical response to internal, residual stresses that accumulate during polymer vitrification. While internal stress is intrinsic to polymers that can form solids, we demonstrate that it can dramatically affect the mechanochemistry of spiropyran probes and alter their intramolecular isomerization barriers by up to 70 kJ mol-1. This new behavior of spiropyrans (SPs) enables their application for analysis of internal stresses distribution and their mechanochemical characterization on the molecular level. Spectroscopy and imaging based on SP mechanochemistry showed high topological sensitivity and allowed us to discern different levels of internal stress impacting various locations along the polymer chain. The nature of the developed technique allows for wide-field imaging of stress heterogeneities in polymer samples of irregular shapes and dimensions, making it feasible to directly observe molecular-level manifestations of mechanical stresses that accompany the formation of a vast number of solid polymers.

Pausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our mechanistic understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high-throughput magnetic tweezers, we examine the Escherichia coli RNAp transcription dynamics over a wide range of forces and NTP concentrations. Dwell-time analysis and stochastic modeling identify, in addition to a short-lived elemental pause, two distinct long-lived backtrack pause states differing in recovery rates. We identify two stochastic sources of transcription heterogeneity: alterations in short-pause frequency that underlies elongation-rate switching, and variations in RNA cleavage rates in long-lived backtrack states. Together with effects of force and Gre factors, we demonstrate that recovery from deep backtracks is governed by intrinsic RNA cleavage rather than diffusional Brownian dynamics. We introduce a consensus mechanistic model that unifies our findings with prior models.

Multiplexed single-molecule magnetic tweezers (MT) have recently been employed to probe the RNA synthesis dynamics of RNA-dependent RNA polymerases (RdRp). Here, we present a protocol for simultaneously probing the RNA synthesis dynamics of hundreds of single polymerases with MT. We describe the preparation of a dsRNA construct for probing single RdRp kinetics. We then detail the measurement of RdRp RNA synthesis kinetics using MT. The protocol is suitable for high-throughput probing of RdRp-targeting antiviral compounds for mechanistic function and efficacy. For complete details on the use and execution of this protocol, please refer to Janissen et al. (2021).

The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ∼50 nm protein complex. Here, using high-resolution magnetic tweezers, we resolve single steps in the loop extrusion process by individual yeast condensins. The measured median step sizes range between 20-40 nm at forces of 1.0-0.2 pN, respectively, comparable with the holocomplex size. These large steps show that, strikingly, condensin typically reels in DNA in very sizeable amounts with ∼200 bp on average per single extrusion step at low force, and occasionally even much larger, exceeding 500 bp per step. Using Molecular Dynamics simulations, we demonstrate that this is due to the structural flexibility of the DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss our findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-binding-induced engagement of the hinge and the globular domain of the SMC complex.

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the “backtracked” state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

Graphene oxide (GO) has immense potential for widespread use in diverse in vitro and in vivo biomedical applications owing to its thermal and chemical resistance, excellent electrical properties and solubility, and high surface-to-volume ratio. However, development of GO-based biological nanocomposites and biosensors has been hampered by its poor intrinsic biocompatibility and difficult covalent biofunctionalization across its lattice. Many studies exploit the strategy of chemically modifying GO by noncovalent and reversible attachment of (bio)molecules or sole covalent biofunctionalization of residual moieties at the lattice edges, resulting in a low coating coverage and a largely bioincompatible composite. Here, we address these problems and present a facile yet powerful method for the covalent biofunctionalization of GO using colamine (CA) and the poly(ethylene glycol) cross-linker that results in a vast improvement in the biomolecular coating density and heterogeneity across the entire GO lattice. We further demonstrate that our biofunctionalized GO with CA as the cross-linker provides superior nonspecific biomolecule adhesion suppression with increased biomarker detection sensitivity in a DNA-biosensing assay compared to the (3-aminopropyl)triethoxysilane cross-linker. Our optimized biofunctionalization method will aid the development of GO-based in situ applications including biosensors, tissue nanocomposites, and drug carriers.

The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptive strategy induced by stresses. In contrast, for diverse human and plant pathogens, filamentous cells have been recently observed during biofilm formation, but their functions and triggering mechanisms remain unclear. To experimentally identify the underlying function and hypothesized cell communication triggers of such cell morphogenesis, spatially controlled cell patterning is pivotal. Here, we demonstrate highly selective cell adhesion of the biofilm-forming phytopathogen Xylella fastidiosa to gold-patterned SiO₂ substrates with well-defined geometries and dimensions. The consequent control of both cell density and distances between cell clusters demonstrated that filamentous cell formation depends on cell cluster density, and their ability to interconnect neighboring cell clusters is distance-dependent. This process allows the creation of large interconnected cell clusters that form the structural framework for macroscale biofilms. The addition of diffusible signaling molecules from supernatant extracts provides evidence that cell filamentation is induced by quorum sensing. These findings and our innovative platform could facilitate therapeutic developments targeting biofilm formation mechanisms of X. fastidiosa and other pathogens.

CpG islands recruit MLL1 via the CXXC domain to modulate chromatin structure and regulate gene expression. The amino acid motif of CXXC also plays a pivotal role in MLL1’s structure and function and serves as a target for drug design. In addition, the CpG pattern in an island governs spatially dependent collaboration among CpGs in recruiting epigenetic enzymes. However, current studies using short DNA fragments cannot probe the dynamics of CXXC on long DNA with crowded CpG motifs. Here, we used single-molecule magnetic tweezers to examine the binding dynamics of MLL1’s CXXC domain on a long DNA with a CpG island. The mechanical strand separation assay allows profiling of protein-DNA complexes and reports force-dependent unfolding times. Further design of a hairpin detector reveals the unfolding time of individual CXXC-CpG complexes. Finally, in a proof of concept we demonstrate the inhibiting effect of dimethyl fumarate on the CXXC-DNA complexes by measuring the dose response curve of the unfolding time. This demonstrates the potential feasibility of using single-molecule strand separation as a label-free detector in drug discovery and chemical biology.

Optical trapping of (sub)micron-sized particles is broadly employed in nanoscience and engineering. The materials commonly employed for these particles, however, have physical properties that limit the transfer of linear or angular momentum (or both). This reduces the magnitude of forces and torques, and the spatiotemporal resolution, achievable in linear and angular traps. Here, we overcome these limitations through the use of single-crystal rutile TiO ₂ , which has an exceptionally large optical birefringence, a high index of refraction, good chemical stability, and is amenable to geometric control at the nanoscale. We show that rutile TiO ₂ nanocylinders form powerful joint force and torque transducers in aqueous environments by using only moderate laser powers to apply nN·nm torques at kHz rotational frequencies to tightly trapped particles. In doing so, we demonstrate how rutile TiO ₂ nanocylinders outperform other materials and offer unprecedented opportunities to expand the control of optical force and torque at the nanoscale.

Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.IMPORTANCE Recombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RNA-dependent RNA polymerase (RdRp) in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, which is indicative of conservation in mechanism.

In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles. Despite markedly condensing the bacterial chromosome, the nucleoid-structuring protein Dps selectively allows access by RNA polymerase and transcription factors at normal rates while excluding other factors such as restriction endonucleases.

Electrically active field-effect transistors (FET) based biosensors are of paramount importance in life science applications, as they offer direct, fast, and highly sensitive label-free detection capabilities of several biomolecules of specific interest. In this work, we report a detailed investigation on surface functionalization and covalent immobilization of biomarkers using biocompatible ethanolamine and poly(ethylene glycol) derivate coatings, as compared to the conventional approaches using silica monoliths, in order to substantially increase both the sensitivity and molecular selectivity of nanowire-based FET biosensor platforms. Quantitative fluorescence, atomic and Kelvin probe force microscopy allowed detailed investigation of the homogeneity and density of immobilized biomarkers on different biofunctionalized surfaces. Significantly enhanced binding specificity, biomarker density, and target biomolecule capture efficiency were thus achieved for DNA as well as for proteins from pathogens. This optimized functionalization methodology was applied to InP nanowires that due to their low surface recombination rates were used as new active transducers for biosensors. The developed devices provide ultrahigh label-free detection sensitivities ∼1 fM for specific DNA sequences, measured via the net change in device electrical resistance. Similar levels of ultrasensitive detection of ∼6 fM were achieved for a Chagas Disease protein marker (IBMP8-1). The developed InP nanowire biosensor provides thus a qualified tool for detection of the chronic infection stage of this disease, leading to improved diagnosis and control of spread. These methodological developments are expected to substantially enhance the chemical robustness, diagnostic reliability, detection sensitivity, and biomarker selectivity for current and future biosensing devices.

Surface attachment of a planktonic bacteria, mediated by adhesins and extracellular polymeric substances (EPS), is a crucial step for biofilm formation. Some pathogens can modulate cell adhesiveness, impacting host colonization and virulence. A framework able to quantify cell-surface interaction forces and their dependence on chemical surface composition may unveil adhesiveness control mechanisms as new targets for intervention and disease control. Here we employed InP nanowire arrays to dissect factors involved in the early stage biofilm formation of the phytopathogen Xylella fastidiosa. Ex vivo experiments demonstrate single-cell adhesion forces up to 45 nN, depending on the cell orientation with respect to the surface. Larger adhesion forces occur at the cell poles; secreted EPS layers and filaments provide additional mechanical support. Significant adhesion force enhancements were observed for single cells anchoring a biofilm and particularly on XadA1 adhesin-coated surfaces, evidencing molecular mechanisms developed by bacterial pathogens to create a stronger holdfast to specific host tissues.