Translation and folding of single proteins in real time.

Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide's vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein's amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein's amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.

Nanoscale Chemical Imaging of Reversible Photoisomerization of an Azobenzene-Thiol Self-Assembled Monolayer by Tip-Enhanced Raman Spectroscopy.

An understanding of the photoisomerization mechanism of molecules bound to a metal surface at the molecular scale is required for designing photoswitches at surfaces. It has remained a challenge to correlate the surface structure and isomerization of photoswitches at ambient conditions. Herein, the photoisomerization of a self-assembled monolayer of azobenzene-thiol molecules on a Au surface was investigated using scanning tunneling microscopy and tip-enhanced Raman spectroscopy. The unique signature of the cis isomer at 1525 cm-1 observed in tip-enhanced Raman spectra was clearly distinct from the trans isomer. Furthermore, tip-enhanced Raman images of azobenzene thiols after ultraviolet and blue light irradiation are shown with nanoscale spatial resolution, demonstrating a reversible conformational change. Interestingly, the cis isomers of azobenzene-thiol molecules were preferentially observed at Au grain edges, which is confirmed by density functional theory.

Microrheological Coagulation Assay Exploiting Micromechanical Resonators.

Rheological measurements in biological liquids yield insights into homeostasis and provide information on important molecular processes that affect fluidity. We present a fully automated cantilever-based method for highly precise and sensitive measurements of microliter sample volumes of human blood plasma coagulation (0.009 cP for viscosity range 0.5-3 cP and 0.0012 g/cm3 for density range 0.9-1.1 g/cm3). Microcantilever arrays are driven by a piezoelectric element, and resonance frequencies and quality factors of sensors that change over time are evaluated. A highly accurate approximation of the hydrodynamic function is introduced that correlates resonance frequency and quality factor of cantilever beams immersed in a fluid to the viscosity and density of that fluid. The theoretical model was validated using glycerol reference solutions. We present a surface functionalization protocol that allows minimization of unspecific protein adsorption onto cantilevers. Adsorption leads to measurement distortions and incorrect estimation of the fluid parameters (viscosity and density). Two hydrophilic terminated self-assembled monolayers (SAMs) sensor surfaces are compared to a hydrophobic terminated SAM coating. As expected, the hydrophobic modified surfaces induced the highest mass adsorption and could promote conformational changes of the proteins and subsequent abnormal biological activity. Finally, the activated partial thromboplastin time (aPTT) coagulation assay was performed, and the viscosity, density, and coagulation rate of human blood plasma were measured along with the standard coagulation time. The method could extend and improve current coagulation testing.

Effect of non-specific species competition from total RNA on the static mode hybridization response of nanomechanical assays of oligonucleotides.

We investigate here the nanomechanical response of microcantilever sensors in real-time for detecting a range of ultra-low concentrations of oligonucleotides in a complex background of total cellular RNA extracts from cell lines without labeling or amplification. Cantilever sensor arrays were functionalized with probe single stranded DNA (ssDNA) and reference ssDNA to obtain a differential signal. They were then exposed to complementary target ssDNA strands that were spiked in a fragmented total cellular RNA background in biologically relevant concentrations so as to provide clinically significant analysis. We present a model for prediction of the sensor behavior in competitive backgrounds with parameters that are indicators of the change in nanomechanical response with variation in the target and background concentration. For nanomechanical assays to compete with current technologies it is essential to comprehend such responses with eventual impact on areas like understanding non-coding RNA pharmacokinetics, nucleic acid biomarker assays and miRNA quantification for disease monitoring and diagnosis to mention a few. Additionally, we also achieved a femtomolar sensitivity limit for online oligonucleotide detection in a non-competitive environment with these sensors.

VirE2: a unique ssDNA-compacting molecular machine.

The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein-VirE2-enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated-using single-molecule techniques-that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.

Quantitative epitope analysis reveals drastic 63% reduced immuno-affinity and 60% enhanced transmissibility for SARS-CoV-2 variants.

SARS-CoV-2 is the cause of a global pandemic that has led to more than 4 million deaths, continues to spread and holds the world in a tight grip. The virus has developed substantial mutations that undermine the efficacy of current vaccines and monoclonal antibody therapies. Semi-quantitative immuno - and neutralization assays are unable to provide direct quantitative insights about the minute variations of emerging mutants. Here, we develop a quantitative assay that enables synchronous screening of emerging variant epitopes with single amino acid resolution. We report on specific label-free quantitative nanomechanical analysis of pseudovirus spike interaction with ACE2 receptors. Within minutes, we can characterize the B.1.1.7 variant transmissibility due to its 63% increased binding, and measure a 60% reduced efficacy of antibodies towards B.1.351 and P.1 variants. Our technology can assist vaccine development studies, with focus on comparing protection patterns and novel vaccine candidates and tracking of immunity over time.

Multimodal real-time frequency tracking of cantilever arrays in liquid environment for biodetection: Comprehensive setup and performance analysis.

We present a nanomechanical platform for real-time quantitative label-free detection of target biomolecules in a liquid environment with mass sensitivity down to few pg. Newly fabricated arrays of up to 18 cantilevers are integrated in a micromachined fluidic chamber, connected to software-controlled fluidic pumps for automated sample injections. We discuss two functionalization approaches to independently sensitize the interface of different cantilevers. A custom piezo-stack actuator and optical readout system enable the measurement of resonance frequencies up to 2 MHz. We implement a new measurement strategy based on a phase-locked loop (PLL), built via in-house developed software. The PLL allows us to track, within the same experiment, the evolution of resonance frequency over time of up to four modes for all the cantilevers in the array. With respect to the previous measurement technique, based on standard frequency sweep, the PLL enhances the estimated detection limit of the device by a factor of 7 (down to 2 pg in 5 min integration time) and the time resolution by more than threefold (below 15 s), being on par with commercial gold-standard techniques. The detection limit and noise of the new setup are investigated via Allan deviation and standard deviation analysis, considering different resonance modes and interface chemistries. As a proof-of-concept, we show the immobilization and label-free in situ detection of live bacterial cells (E. coli), demonstrating qualitative and quantitative agreement in the mechanical response of three different resonance modes.

Nanotechnological immunoassay for rapid label-free analysis of candidate malaria vaccines.

Malaria is a life-threatening epidemic disease with half of the world's population at risk. Although its incidence rate has fallen since 2010, this ratio dramatically stalled between 2014 and 2018. New fast and optimized tools in vaccine analysis and seroconversion testing are critically needed. We developed a clinical diagnostic device based on piezo-actuated nanoresonators that perform as quantitative in situ calibrated nano-bio sensors for specific detection of multiple target molecules in serum samples. The immunoassay successfully diagnoses humoral immune responses induced by malaria vaccine candidates and reveals the timeline and stage of the infection. We applied the newly developed strategy to a variety of different samples, from pure antibody/vaccine solutions, to blood samples from clinical trials on both naïve and pre-exposed malaria volunteers from sub-Saharan countries. Our nanomechanical assay provides a direct one-step label-free quantitative immunoassay that is on par with the gold-standard, multi-step enzyme-linked immunosorbent assay (ELISA). We achieve a limit of detection of few pg ml-1, or sub-pM concentrations. The 6 µl sample volume allows more than 50 experiments from one finger prick. Furthermore, we simultaneously detected multiple analytes by differential functionalization of multiple sensors in parallel. The inherent differential read-out with in situ controls reduces false positive results. Due to the faster turnaround time, the minimal volume required and the automatized handling system, this technique has great potential for miniaturization and routine diagnostics in pandemic emergencies.

Fast quantitative single-molecule detection at ultralow concentrations.

The applicability of single-molecule fluorescence assays in liquids is limited by diffusion to concentrations in the low picomolar range. Here, we demonstrate quantitative single-molecule detection at attomolar concentrations within 1 min by excitation and detection of fluorescence through a single-mode optical fiber in presence of turbulent flow. The combination of high detectability and short measurement times promises applications in ultrasensitive assays, sensors, and point-of-care medical diagnostics.

Interaction of cationic surfactants with DNA: a single-molecule study.

The interaction of cationic surfactants with single dsDNA molecules has been studied using force-measuring optical tweezers. For hydrophobic chains of length 12 and greater, pulling experiments show characteristic features (e.g. hysteresis between the pulling and relaxation curves, force-plateau along the force curves), typical of a condensed phase (compaction of a long DNA into a micron-sized particle). Depending on the length of the hydrophobic chain of the surfactant, we observe different mechanical behaviours of the complex (DNA-surfactants), which provide evidence for different binding modes. Taken together, our measurements suggest that short-chain surfactants, which do not induce any condensation, could lie down on the DNA surface and directly interact with the DNA grooves through hydrophobic-hydrophobic interactions. In contrast, long-chain surfactants could have their aliphatic tails pointing away from the DNA surface, which could promote inter-molecular interactions between hydrophobic chains and subsequently favour DNA condensation.

Detection of transient events in the presence of background noise.

We describe a method to detect and count transient burstlike signals in the presence of a significant stationary noise. To discriminate a transient signal from the background noise, an optimum threshold is determined using an iterative algorithm that yields the probability distribution of the background noise. Knowledge of the probability distribution of the noise then allows the determination of the number of transient events with a quantifiable error (wrong-positives). We apply the method, which does not rely on the choice of free parameters, to the detection and counting of transient single-molecule fluorescence events in the presence of a strong background noise. The method will be of importance in various ultra sensing applications.

Towards personalised rapid label free miRNA detection for cancer and liver injury diagnostics in cell lysates and blood based samples.

Advances in prevention, diagnosis and therapy are coupled to innovation and development of new medical tools, leading to improved patient prognosis. We developed an automatic biosensor platform that could provide a non-invasive, rapid and personalised diagnosis using nanomechanical cantilever sensors. miRNA are involved in gene expression and are extractable biomarkers for multiple diseases. We detected specific expression patterns of miRNA relevant to cancer and adverse drug effects directly in cell lysates or blood based samples using only a few microliters of sample within one hour. Specific miRNA hybridisation to the upper cantilever surface induces physical bending of the sensor which is detected by monitoring the position of a laser that reflects from the sensors surface. Internal reference sensors negate environmental and nonspecific effects. We showed that the sensitivity of label free cantilever nanomechanical sensing of miRNA surpasses that of surface plasmon resonance by more than three orders of magnitude. A cancer associated miRNA expression profile from cell lysates and one associated with hepatocytes derived from necrotic liver tissue in blood-based samples has been successfully detected. Our label free mechanical approach displays the capability to perform in relevant clinical samples while also obtaining comparable results to PCR based techniques. Without the need to individually extend, amplify or label each target allowing multitarget analysis from one sample.

Nanomechanical clinical coagulation diagnostics and monitoring of therapies.

Clinical coagulation diagnostics often requires multiple tests. Coagulation times are a first indication of an abnormal coagulation process, such as a coagulation factor deficiency. To determine the specific deficient factor, additional immuno- and/or enzyme assays are necessary. Currently, every clinical laboratory has to normalize their assays (international normalized ratio, INR), and therefore, certain variability within the clinical analytics exists. We report a novel strategy for a quick, reliable and quantitative diagnosis of blood coagulation diseases (e.g. haemophilia) and for monitoring factor replacement and anticoagulant therapies (e.g. heparin treatment). We exploit nano-oscillations of microcantilevers for real-time measurements of the evolving blood plasma clot strength (viscosity). The sensors are oscillated at multiple high resonance mode numbers, in order to minimise the oscillation amplitude (a few nanometers), to provide direct internal control and to increase the quality factor. Along with the activated thromboplastin time (aPTT) and prothrombin time (PT) other parameters important for thrombosis diagnostics can be obtained, including the final clot strength and the fibrinolysis time. We demonstrate the dependence of the parameters on factor deficiencies and we diagnose a specific factor deficiency through an integrated and quantitative in situ immunoassay. This approach does not require continuous calibration since it delivers an absolute quantity (clot strength). The low sample volume required (a few µl) and the ability to measure different parameters within the same test (PT, aPTT and global coagulation assay) make the presented technique a versatile point-of-care device for clinical coagulation diagnostics.

Myomesin is a molecular spring with adaptable elasticity.

The M-band is a transverse structure in the center of the sarcomere, which is thought to stabilize the thick filament lattice. It was shown recently that the constitutive vertebrate M-band component myomesin can form antiparallel dimers, which might cross-link the neighboring thick filaments. Myomesin consists mainly of immunoglobulin-like (Ig) and fibronectin type III (Fn) domains, while several muscle types express the EH-myomesin splice isoform, generated by the inclusion of the unique EH-segment of about 100 amino acid residues (aa) in the center of the molecule. Here we use atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy for the biophysical characterization of myomesin. The AFM identifies the "mechanical fingerprints" of the modules constituting the myomesin molecule. Stretching of homomeric polyproteins, constructed of Ig and Fn domains of human myomesin, produces a typical saw-tooth pattern in the force-extension curve. The domains readily refold after relaxation. In contrast, stretching of a heterogeneous polyprotein, containing several repeats of the My6-EH fragment reveals a long initial plateau corresponding to the sum of EH-segment contour lengths, followed by several My6 unfolding peaks. According to this, the EH-segment is characterized as an entropic chain with a persistence length of about 0.3nm. In TEM pictures, the EH-domain appears as a gap in the molecule, indicating a random coil conformation similar to the PEVK region of titin. CD spectroscopy measurements support this result, demonstrating a mostly non-folded conformation for the EH-segment. We suggest that similarly to titin, myomesin is a molecular spring, whose elasticity is modulated by alternative splicing. The Ig and Fn domains might function as reversible "shock absorbers" by sequential unfolding in the case of extremely high or long sustained stretching forces. These complex visco-elastic properties of myomesin might be crucial for the stability of the sarcomere.

Label free analysis of transcription factors using microcantilever arrays.

We report the measurement of protein interaction with double-stranded DNA oligonucleotides using cantilever microarray technology. We investigated two different DNA-binding proteins, the transcription factors SP1 and NF-kappaB, using cantilever arrays as they allow label-free measurement of different biomolecular interactions in parallel. Double-stranded DNA oligonucleotides containing a specific binding site for a transcription factor were sensitized on gold-coated cantilevers. The binding of the transcription factor creates a surface stress, resulting in a bending of the cantilevers. Both transcription factors could be detected independently at concentrations of 80-100 nM. A concentration dependence of the bending signal was measured using concentrations from 100 to 400 nM of NF-kappaB. The experiments show that the recognition sequence of one transcription factor can serve as a reference for the other, highlighting the sequence specificity of transcription factor binding.

Single-molecule mechanics of protein-labelled DNA handles.

DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA-protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot-streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein-DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time-critical molecular motor studies.

Micromechanical oscillators as rapid biosensor for the detection of active growth of Escherichia coli.

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated by common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The sensor is able to detect active growth of Escherichia coli cells within 1 h which is significantly faster than any conventional plating method which requires at least 24 h. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of active bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing by adding antibiotics to the nutritive layer.

Micromechanical cantilever array sensors for selective fungal immobilization and fast growth detection.

We demonstrate the use of micromechanical cantilever arrays for selective immobilization and fast quantitative detection of vital fungal spores. Micro-fabricated uncoated as well as gold-coated silicon cantilevers were functionalized with concanavalin A, fibronectin or immunoglobulin G. In our experiments two major morphological fungal forms were used--the mycelial form Aspergillus niger and the unicellular yeast form Saccharomyces cerevisiae, as models to explore a new method for growth detection of eukaryotic organisms using cantilever arrays. We exploited the specific biomolecular interactions of surface grafted proteins with the molecular structures on the fungal cell surface. It was found that these proteins have different affinities and efficiencies to bind the spores. Maximum spore immobilization, germination and mycelium growth was observed on the immunoglobulin G functionalized cantilever surfaces. We show that spore immobilization and germination of the mycelial fungus A. niger and yeast S. cerevisiae led to shifts in resonance frequency within a few hours as measured by dynamically operated cantilever arrays, whereas conventional techniques would require several days. The biosensor could detect the target fungi in a range of 10(3) - 10(6) CFUml(-1). The measured shift is proportional to the mass of single fungal spores and can be used to evaluate spore contamination levels. Applications lie in the field of medical and agricultural diagnostics, food- and water-quality monitoring.

Micromechanical mass sensors for biomolecular detection in a physiological environment.

Micromechanical cantilever arrays are used to measure time-resolved adsorption of tiny masses based on protein-ligand interactions. Here, streptavidin-biotin interactions are investigated in a physiological environment. A measurement method is introduced using higher flexural modes of a silicon cantilever in order to enhance the sensitivity of mass detection. Modeling the cantilever vibration in liquid allows the measurement of absolute mass changes. We show time-resolved mass adsorption of final 7+/-0.7 ng biotinylated latex beads. The sensitivity obtained is about 2.5 pg/Hz measuring at a center frequency of 750 kHz.

On-surface derivatisation of aromatic molecules on graphene: the importance of packing density.

An efficient, high-throughput method for the formation of densely packed molecular films on graphene is reported. The films exhibit high stability and remain intact during a subsequent derivatisation reaction, offering a versatile route for the non-covalent functionalisation of graphene.