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Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., BN/Chirlmin Joo Lab, Sports & Games, BN/Bionanoscience

The inherent properties of 2D materials—light mass, high out-of-plane flexibility, and large surface area—promise great potential for precise and accurate nanomechanical mass sensing, but their application is often hampered by surface contamination. Here we demonstrate a tri-layer graphene nanomechanical resonant mass sensor with sub-attogram resolution at room temperature, fabricated by a bottom-up process. We found that Joule-heating is effective in cleaning the graphene membrane surface, which results in a large improvement in the stability of the resonance frequency. We characterized the sensor by depositing Cr metal using a stencil mask and found a mass-resolution that is sufficient to weigh very small particles, like large proteins and protein complexes, with potential applications in the fields of nanobiology and medicine., Dynamics of Micro and Nano Systems, BN/Chirlmin Joo Lab

Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon that has been repurposed as a biophysical tool to measure nanometer distances. With FRET by DNA eXchange, or FRET X, many points of interest (POIs) in a single object can be probed, overcoming a major limitation of conventional single-molecule FRET. In FRET X, short fluorescently labeled DNA imager strands specifically and transiently bind their complementary docking strands on a target molecule, such that at most a single FRET pair is formed at each point in time and multiple POIs on a single molecule can be readily probed. Here, we describe the sample preparation, image acquisition, and data analysis for structural analysis of DNA nanostructures with FRET X., Green Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., BN/Chirlmin Joo Lab

Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity and kinetics measurements enabled the construction of energy landscapes in sequence space, uncovering relationships between sequence, structure, and function. Here, we review the approaches to perform ensemble fluorescence experiments on next-generation sequencing chips for variations of DNA, RNA, and protein sequences. As the next step, we anticipate that these fluorescence experiments will be pushed to the single-molecule level, which can directly uncover kinetics and molecular heterogeneity in an unprecedented high-throughput fashion. Molecular biophysics in sequence space, both at the ensemble and single-molecule level, leads to new mechanistic insights. The wide spectrum of applications in biology and medicine ranges from the fundamental understanding of evolutionary pathways to the development of new therapeutics., Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., BN/Bionanoscience, BN/Chirlmin Joo Lab

Nanomechanical resonator devices are widely used as ultrasensitive mass detectors for fundamental studies and practical applications. The resonance frequency of the resonators shifts when a mass is loaded, which is used to estimate the mass. However, the shift signal is often blurred by the thermal noise, which interferes with accurate mass detection. Here, we demonstrate the reduction of the noise interference in mass detection in suspended graphene-based nanomechanical resonators, by using applied machine learning. Featurization is divided into image and sequential datasets, and those datasets are trained and classified using 2D and 1D convolutional neural networks (CNNs). The 2D CNN learning-based classification shows a performance with f1-score over 99% when the resonance frequency shift is more than 2.5% of the amplitude of the thermal noise range., Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., BN/Chirlmin Joo Lab

In recent years, single molecule technology has experienced a rapid growth, with exciting developments in fundamental research and real-world applications. Detecting and studying biological phenomena on the single molecule level requires a unique synergy between researchers working on instrumentation, physics, and the life sciences. In the iScience special issue ‘‘Single Molecule Technology – From Biotechnology to Biomedical Applications’’, guest edited by Amit Meller and Chirlmin Joo (Figure 1), we are highlighting a variety of research on nanopore technology, single molecule fluorescence, and a selection of other ultra- sensitive detection methods. More content in the special issue can be found here: https://www.sciencedirect.com/journal/iscience/special-issue/10PGSBV55N0. The guest editors in this backstory share their thoughts on what is currently exciting in the field, and the advances they think will make an impact in the near future., BN/Chirlmin Joo Lab

Single-molecule protein identification is an unrealized concept with potentially ground-breaking applications in biological research. We propose a method called FRET X (Förster Resonance Energy Transfer via DNA eXchange) fingerprinting, in which the FRET efficiency is read out between exchangeable dyes on protein-bound DNA docking strands and accumulated FRET efficiencies constitute the fingerprint for a protein. To evaluate the feasibility of this approach, we simulated fingerprints for hundreds of proteins using a coarse-grained lattice model and experimentally demonstrated FRET X fingerprinting on model peptides. Measured fingerprints are in agreement with our simulations, corroborating the validity of our modeling approach. In a simulated complex mixture of >300 human proteins of which only cysteines, lysines, and arginines were labeled, a support vector machine was able to identify constituents with 95% accuracy. We anticipate that our FRET X fingerprinting approach will form the basis of an analysis tool for targeted proteomics., BN/Chirlmin Joo Lab

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics., Accepted Author Manuscript, ChemE/Advanced Soft Matter, BN/Chirlmin Joo Lab, BN/Cees Dekker Lab

Localization microscopy offers resolutions down to a single nanometer but currently requires additional dedicated hardware or fiducial markers to reduce resolution loss from the drift of the sample. Drift estimation without fiducial markers is typically implemented using redundant cross correlation (RCC). We show that RCC has sub-optimal precision and bias, which leaves room for improvement. Here, we minimize a bound on the entropy of the obtained localizations to efficiently compute a precise drift estimate. Within practical compute-time constraints, simulations show a 5x improvement in drift estimation precision over the widely used RCC algorithm. The algorithm operates directly on fluorophore localizations and is tested on simulated and experimental datasets in 2D and 3D. An open source implementation is provided, implemented in Python and C++, and can utilize a GPU if available., Team Carlas Smith, BN/Chirlmin Joo Lab

Single-molecule techniques allow the visualization of the molecular dynamics of nucleic acids and proteins with high spatiotemporal resolution. Valuable kinetic information of biomolecules can be obtained when the discrete states within single-molecule time trajectories are determined. Here, we present a fast, automated, and bias-free step detection method, AutoStepfinder, that determines steps in large datasets without requiring prior knowledge on the noise contributions and location of steps. The analysis is based on a series of partition events that minimize the difference between the data and the fit. A dual-pass strategy determines the optimal fit and allows AutoStepfinder to detect steps of a wide variety of sizes. We demonstrate step detection for a broad variety of experimental traces. The user-friendly interface and the automated detection of AutoStepfinder provides a robust analysis procedure that enables anyone without programming knowledge to generate step fits and informative plots in less than an hour., BN/Chirlmin Joo Lab, BN/Technici en Analisten, BN/Cees Dekker Lab

Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented control on targeting and editing specific DNA sequences. If the target sequences are prone to folding into noncanonical secondary structures, such as G-quadruplex (GQ), the conformational states and activity of the CRISPR-Cas9 complex may be influenced, but the impact has not been assessed. Using single molecule FRET, we investigated structural characteristics of the complex formed by CRISPR-Cas9 and target DNA, which contains a potentially GQ forming sequence (PQS) in either the target or the nontarget strand (TS or NTS). We observed different conformational states and dynamics depending on the stability of the GQ and the position of PQS. When PQS was in NTS, we observed evidence for GQ formation for both weak and stable GQs. This is consistent with R-loop formation between TS and crRNA releasing NTS from Watson-Crick pairing and facilitating secondary structure formation in it. When PQS was in TS, R-loop formation was adequate to maintain a weak GQ in the unfolded state but not a GQ with moderate or high stability. The observed structural heterogeneity within the target dsDNA and the R-loop strongly depended on whether the PQS was in TS or NTS. We propose these variations in the complex structures to have functional implications for Cas9 activity., BN/Chirlmin Joo Lab

Transposable elements have both detrimental and beneficial effects on their host genome. Tetrahymena is a unicellular eukaryote that deals with transposable elements in a unique way. It has a separate somatic and germline genome in two nuclei in a single cell. During sexual reproduction, a small RNA directed system compares the germline and somatic genome to identify transposable elements and related sequences. These are subsequently marked by heterochromatin and excised. In this Review, current knowledge of this system and the gaps therein are discussed. Additionally, the possibility to exploit the Tetrahymena machinery for genome editing and its advantages over the widely used CRISPR-Cas9 system will be explored. While the bacterial derived CRISPR-Cas9 has difficulty to access eukaryotic chromatin, Tetrahymena proteins are adept at acting in a chromatin context. Furthermore, Tetrahymena based gene therapy in humans might be a safer alternative to Cas9 because the latter can trigger an immune response., BN/Chirlmin Joo Lab

Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object, which makes it difficult to apply single-molecule FRET for structural analysis of biomolecules. Here, we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the pair distance with subnanometer precision. The distance between other pairs are determined by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. Our FRET X technology will be a tool for the high-resolution analysis of biomolecules and nanostructures., BN/Chirlmin Joo Lab

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever- increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB- Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices., BN/Chirlmin Joo Lab

Single-molecule localization microscopy (SMLM) is a potent tool to examine biological systems with unprecedented resolution, enabling the investigation of increasingly smaller structures. At the forefront of these developments is DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), which exploits the stochastic and transient binding of fluorescently labeled DNA probes. In its early stages the implementation of DNA-PAINT was burdened by low-throughput, excessive acquisition time, and difficult integration with live-cell imaging. However, recent advances are addressing these challenges and expanding the range of applications of DNA-PAINT. We review the current state of the art of DNA-PAINT in light of these advances and contemplate what further developments remain indispensable to realize live-cell imaging., Accepted Author Manuscript, BN/Chirlmin Joo Lab

Förster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nanoscale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner but lack the flexibility to adapt to different experimental setups and require local installations. Here, we propose to fit models to optical signals more intuitively by adopting a semisupervised approach, in which the user interactively guides the model to fit a given data set, and introduce FRETboard, a web tool that allows users to provide such guidance. We show that our approach is able to closely reproduce ground truth FRET statistics in a wide range of simulated single-molecule scenarios and correctly estimate parameters for up to 11 states. On in vitro data, we retrieve parameters identical to those obtained by laborious manual classification in a fraction of the required time. Moreover, we designed FRETboard to be easily extendable to other models, allowing it to adapt to future developments in FRET measurement and analysis., Accepted Author Manuscript, BN/Chirlmin Joo Lab

Single-molecule protein identification is an unrealized concept with potentially ground-breaking applications in biological research. We propose a method called FRET X (Förster Resonance Energy Transfer via DNA eXchange) fingerprinting, in which the FRET efficiency is read out between exchangeable dyes on protein-bound DNA docking strands and accumulated FRET efficiencies constitute the fingerprint for a protein. To evaluate the feasibility of this approach, we simulated fingerprints for hundreds of proteins using a coarse-grained lattice model and experimentally demonstrated FRET X fingerprinting on model peptides. Measured fingerprints are in agreement with our simulations, corroborating the validity of our modeling approach. In a simulated complex mixture of >300 human proteins of which only cysteines, lysines, and arginines were labeled, a support vector machine was able to identify constituents with 95% accuracy. We anticipate that our FRET X fingerprinting approach will form the basis of an analysis tool for targeted proteomics., BN/Chirlmin Joo Lab

In recent years, single molecule technology has experienced a rapid growth, with exciting developments in fundamental research and real-world applications. Detecting and studying biological phenomena on the single molecule level requires a unique synergy between researchers working on instrumentation, physics, and the life sciences. In the iScience special issue ‘‘Single Molecule Technology – From Biotechnology to Biomedical Applications’’, guest edited by Amit Meller and Chirlmin Joo (Figure 1), we are highlighting a variety of research on nanopore technology, single molecule fluorescence, and a selection of other ultra- sensitive detection methods. More content in the special issue can be found here: https://www.sciencedirect.com/journal/iscience/special-issue/10PGSBV55N0. The guest editors in this backstory share their thoughts on what is currently exciting in the field, and the advances they think will make an impact in the near future., BN/Chirlmin Joo Lab

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics., Accepted Author Manuscript, ChemE/Advanced Soft Matter, BN/Chirlmin Joo Lab, BN/Cees Dekker Lab

Localization microscopy offers resolutions down to a single nanometer but currently requires additional dedicated hardware or fiducial markers to reduce resolution loss from the drift of the sample. Drift estimation without fiducial markers is typically implemented using redundant cross correlation (RCC). We show that RCC has sub-optimal precision and bias, which leaves room for improvement. Here, we minimize a bound on the entropy of the obtained localizations to efficiently compute a precise drift estimate. Within practical compute-time constraints, simulations show a 5x improvement in drift estimation precision over the widely used RCC algorithm. The algorithm operates directly on fluorophore localizations and is tested on simulated and experimental datasets in 2D and 3D. An open source implementation is provided, implemented in Python and C++, and can utilize a GPU if available., Team Carlas Smith, BN/Chirlmin Joo Lab

Single-molecule techniques allow the visualization of the molecular dynamics of nucleic acids and proteins with high spatiotemporal resolution. Valuable kinetic information of biomolecules can be obtained when the discrete states within single-molecule time trajectories are determined. Here, we present a fast, automated, and bias-free step detection method, AutoStepfinder, that determines steps in large datasets without requiring prior knowledge on the noise contributions and location of steps. The analysis is based on a series of partition events that minimize the difference between the data and the fit. A dual-pass strategy determines the optimal fit and allows AutoStepfinder to detect steps of a wide variety of sizes. We demonstrate step detection for a broad variety of experimental traces. The user-friendly interface and the automated detection of AutoStepfinder provides a robust analysis procedure that enables anyone without programming knowledge to generate step fits and informative plots in less than an hour., BN/Chirlmin Joo Lab, BN/Technici en Analisten, BN/Cees Dekker Lab

Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object, which makes it difficult to apply single-molecule FRET for structural analysis of biomolecules. Here, we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the pair distance with subnanometer precision. The distance between other pairs are determined by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. Our FRET X technology will be a tool for the high-resolution analysis of biomolecules and nanostructures., BN/Chirlmin Joo Lab

Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented control on targeting and editing specific DNA sequences. If the target sequences are prone to folding into noncanonical secondary structures, such as G-quadruplex (GQ), the conformational states and activity of the CRISPR-Cas9 complex may be influenced, but the impact has not been assessed. Using single molecule FRET, we investigated structural characteristics of the complex formed by CRISPR-Cas9 and target DNA, which contains a potentially GQ forming sequence (PQS) in either the target or the nontarget strand (TS or NTS). We observed different conformational states and dynamics depending on the stability of the GQ and the position of PQS. When PQS was in NTS, we observed evidence for GQ formation for both weak and stable GQs. This is consistent with R-loop formation between TS and crRNA releasing NTS from Watson-Crick pairing and facilitating secondary structure formation in it. When PQS was in TS, R-loop formation was adequate to maintain a weak GQ in the unfolded state but not a GQ with moderate or high stability. The observed structural heterogeneity within the target dsDNA and the R-loop strongly depended on whether the PQS was in TS or NTS. We propose these variations in the complex structures to have functional implications for Cas9 activity., BN/Chirlmin Joo Lab

Transposable elements have both detrimental and beneficial effects on their host genome. Tetrahymena is a unicellular eukaryote that deals with transposable elements in a unique way. It has a separate somatic and germline genome in two nuclei in a single cell. During sexual reproduction, a small RNA directed system compares the germline and somatic genome to identify transposable elements and related sequences. These are subsequently marked by heterochromatin and excised. In this Review, current knowledge of this system and the gaps therein are discussed. Additionally, the possibility to exploit the Tetrahymena machinery for genome editing and its advantages over the widely used CRISPR-Cas9 system will be explored. While the bacterial derived CRISPR-Cas9 has difficulty to access eukaryotic chromatin, Tetrahymena proteins are adept at acting in a chromatin context. Furthermore, Tetrahymena based gene therapy in humans might be a safer alternative to Cas9 because the latter can trigger an immune response., BN/Chirlmin Joo Lab

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever- increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB- Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices., BN/Chirlmin Joo Lab

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription., BN/Chirlmin Joo Lab

Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions., BN/Chirlmin Joo Lab, BN/Cees Dekker Lab, BN/Technici en Analisten

Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions., BN/Chirlmin Joo Lab, BN/Cees Dekker Lab, BN/Technici en Analisten

Supramolecular encapsulation is known to alter chemical properties of guest molecules. We have applied this strategy of molecular encapsulation to temporally control the catalytic activity of a stable copper(I)–carbene catalyst. Encapsulation of the copper(I)–carbene catalyst by the supramolecular host cucurbit[7]uril (CB[7]) resulted in the complete inactivation of a copper-catalyzed alkyne–azide cycloaddition (CuAAC) reaction. The addition of a chemical signal achieved the near instantaneous activation of the catalyst, by releasing the catalyst from the inhibited CB[7] catalyst complex. To broaden the scope of our on-demand CuAAC reaction, we demonstrated the protein labeling of vinculin with the copper(I)–carbene catalyst, to inhibit its activity by encapsulation with CB[7] and to initiate labeling at any moment by adding a specific signal molecule. Ultimately, this strategy allows for temporal control over copper-catalyzed click chemistry, on small molecules as well as protein targets., ChemE/Advanced Soft Matter, BN/Chirlmin Joo Lab, BT/Biocatalysis

Supramolecular encapsulation is known to alter chemical properties of guest molecules. We have applied this strategy of molecular encapsulation to temporally control the catalytic activity of a stable copper(I)–carbene catalyst. Encapsulation of the copper(I)–carbene catalyst by the supramolecular host cucurbit[7]uril (CB[7]) resulted in the complete inactivation of a copper-catalyzed alkyne–azide cycloaddition (CuAAC) reaction. The addition of a chemical signal achieved the near instantaneous activation of the catalyst, by releasing the catalyst from the inhibited CB[7] catalyst complex. To broaden the scope of our on-demand CuAAC reaction, we demonstrated the protein labeling of vinculin with the copper(I)–carbene catalyst, to inhibit its activity by encapsulation with CB[7] and to initiate labeling at any moment by adding a specific signal molecule. Ultimately, this strategy allows for temporal control over copper-catalyzed click chemistry, on small molecules as well as protein targets., ChemE/Advanced Soft Matter, BN/Chirlmin Joo Lab, BT/Biocatalysis

Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions., BN/Chirlmin Joo Lab, BN/Cees Dekker Lab, BN/Technici en Analisten

Post-translational modifications (PTMs) of proteins play key roles in cellular processes. Hence, PTM identification is crucial for elucidating the mechanism of complex cellular processes and disease. Here we present a method for PTM detection at the single-molecule level using FraC biological nanopores. We focus on two major PTMs, phosphorylation and glycosylation, that mutually compete for protein modification sites, an important regulatory process that has been implicated in the pathogenic pathways of many diseases. We show that phosphorylated and glycosylated peptides can be clearly differentiated from nonmodified peptides by differences in the relative current blockade and dwell time in nanopore translocations. Furthermore, we show that these PTM modifications can be mutually differentiated, demonstrating the identification of phosphorylation and glycosylation in a label-free manner. The results represent an important step for the single-molecule, label-free identification of proteoforms, which have tremendous potential for disease diagnosis and cell biology., BN/Chirlmin Joo Lab, BUS/Quantum Delft, BN/Cees Dekker Lab

While DNA sequencing is now amply available, fast, and inexpensive, protein sequencing remains a tremendous challenge. Nanopores may allow for developing a protein sequencer with single-molecule capabilities. As identification of 20 different amino acids currently presents an unsurmountable challenge, fingerprinting schemes are pursued, in which only a subset of amino acids is labeled and detected. This requires modification of amino acids with chemical structures that generate a distinct nanopore ionic current signal. Here, we use a model peptide and the fragaceatoxin C nanopore to characterize six potential tags for a fingerprinting approach using nanopores. We find that labeled and unlabeled proteins can be clearly distinguished and that sensitive detection is obtained for labels with a spectrum of different physicochemical properties such as mass (427-1275 Da), geometry, charge, and hydrophobicity. Additionally, information about the position of the label along the peptide chain can be obtained from individual current-blockade event features. The results represent an important advance toward the development of a single-molecule protein-fingerprinting device with nanopores., BN/Chirlmin Joo Lab, BN/Christophe Danelon Lab, ChemE/Advanced Soft Matter, BN/Cees Dekker Lab

Argonaute (Ago) proteins are of key importance in many cellular processes. In eukaryotes, Ago can induce translational repression followed by deadenylation and degradation of mRNA molecules through base pairing of microRNAs (miRNAs) with a complementary target on a mRNA sequence. In bacteria, Ago eliminates foreign DNA through base pairing of siDNA (small interfering DNA) with a target on a DNA sequence. Effective targeting activities of Ago require fast recognition of the cognate target sequence among numerous off-target sites. Other target search proteins such as transcription factors (TFs) are known to rely on facilitated diffusion for this goal, but it is undetermined to what extent these small nucleic acid-guided proteins utilize this mechanism. Here, we review recent single-molecule studies on Ago target search. We discuss the consequences of the recent findings on the search mechanism. Furthermore, we discuss the open standing research questions that need to be addressed for a complete picture of facilitated target search by small nucleic acids., BN/Chirlmin Joo Lab

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. Using single-molecule FRET in conjunction with kinetic modelling, we reveal that target scanning is mediated through loose protein-nucleic acid interactions, allowing Ago to slide short distances over secondary structures, as well as to bypass protein barriers via intersegmental transfer. Our combined single-molecule experiment and kinetic modelling approach may serve as a platform to dissect search processes and study the effect of sequence on search kinetics for other nucleic acid-guided proteins., BN/Chirlmin Joo Lab, BN/Martin Depken Lab

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications., BN/Chirlmin Joo Lab

Ion channels form the basis of information processing in living cells by facilitating the exchange of electrical signals across and along cellular membranes. Applying the same principles to man-made systems requires the development of synthetic ion channels that can alter their conductance in response to a variety of external manipulations. By combining single-molecule electrical recordings with all-atom molecular dynamics simulations, we here demonstrate a hybrid nanopore system that allows for both a stepwise change of its conductance and a nonlinear current-voltage dependence. The conductance modulation is realized by using a short flexible peptide gate that carries opposite electric charge at its ends. We show that a constant transmembrane bias can position (and, in a later stage, remove) the peptide gate right at the most-sensitive sensing region of a biological nanopore FraC, thus partially blocking its channel and producing a stepwise change in the conductance. Increasing or decreasing the bias while having the peptide gate trapped in the pore stretches or compresses the peptide within the nanopore, thus modulating its conductance in a nonlinear but reproducible manner. We envision a range of applications of this removable-gate nanopore system, e.g. from an element of biological computing circuits to a test bed for probing the elasticity of intrinsically disordered proteins., Accepted Author Manuscript, BN/Chirlmin Joo Lab, BN/Cees Dekker Lab

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. Using single-molecule FRET in conjunction with kinetic modelling, we reveal that target scanning is mediated through loose protein-nucleic acid interactions, allowing Ago to slide short distances over secondary structures, as well as to bypass protein barriers via intersegmental transfer. Our combined single-molecule experiment and kinetic modelling approach may serve as a platform to dissect search processes and study the effect of sequence on search kinetics for other nucleic acid-guided proteins., BN/Chirlmin Joo Lab, BN/Martin Depken Lab

Post-translational modifications (PTMs) of proteins play key roles in cellular processes. Hence, PTM identification is crucial for elucidating the mechanism of complex cellular processes and disease. Here we present a method for PTM detection at the single-molecule level using FraC biological nanopores. We focus on two major PTMs, phosphorylation and glycosylation, that mutually compete for protein modification sites, an important regulatory process that has been implicated in the pathogenic pathways of many diseases. We show that phosphorylated and glycosylated peptides can be clearly differentiated from nonmodified peptides by differences in the relative current blockade and dwell time in nanopore translocations. Furthermore, we show that these PTM modifications can be mutually differentiated, demonstrating the identification of phosphorylation and glycosylation in a label-free manner. The results represent an important step for the single-molecule, label-free identification of proteoforms, which have tremendous potential for disease diagnosis and cell biology., BN/Chirlmin Joo Lab, BUS/Quantum Delft, BN/Cees Dekker Lab

While DNA sequencing is now amply available, fast, and inexpensive, protein sequencing remains a tremendous challenge. Nanopores may allow for developing a protein sequencer with single-molecule capabilities. As identification of 20 different amino acids currently presents an unsurmountable challenge, fingerprinting schemes are pursued, in which only a subset of amino acids is labeled and detected. This requires modification of amino acids with chemical structures that generate a distinct nanopore ionic current signal. Here, we use a model peptide and the fragaceatoxin C nanopore to characterize six potential tags for a fingerprinting approach using nanopores. We find that labeled and unlabeled proteins can be clearly distinguished and that sensitive detection is obtained for labels with a spectrum of different physicochemical properties such as mass (427-1275 Da), geometry, charge, and hydrophobicity. Additionally, information about the position of the label along the peptide chain can be obtained from individual current-blockade event features. The results represent an important advance toward the development of a single-molecule protein-fingerprinting device with nanopores., BN/Chirlmin Joo Lab, BN/Christophe Danelon Lab, ChemE/Advanced Soft Matter, BN/Cees Dekker Lab

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications., BN/Chirlmin Joo Lab

Argonaute (Ago) proteins are of key importance in many cellular processes. In eukaryotes, Ago can induce translational repression followed by deadenylation and degradation of mRNA molecules through base pairing of microRNAs (miRNAs) with a complementary target on a mRNA sequence. In bacteria, Ago eliminates foreign DNA through base pairing of siDNA (small interfering DNA) with a target on a DNA sequence. Effective targeting activities of Ago require fast recognition of the cognate target sequence among numerous off-target sites. Other target search proteins such as transcription factors (TFs) are known to rely on facilitated diffusion for this goal, but it is undetermined to what extent these small nucleic acid-guided proteins utilize this mechanism. Here, we review recent single-molecule studies on Ago target search. We discuss the consequences of the recent findings on the search mechanism. Furthermore, we discuss the open standing research questions that need to be addressed for a complete picture of facilitated target search by small nucleic acids., BN/Chirlmin Joo Lab

Ion channels form the basis of information processing in living cells by facilitating the exchange of electrical signals across and along cellular membranes. Applying the same principles to man-made systems requires the development of synthetic ion channels that can alter their conductance in response to a variety of external manipulations. By combining single-molecule electrical recordings with all-atom molecular dynamics simulations, we here demonstrate a hybrid nanopore system that allows for both a stepwise change of its conductance and a nonlinear current-voltage dependence. The conductance modulation is realized by using a short flexible peptide gate that carries opposite electric charge at its ends. We show that a constant transmembrane bias can position (and, in a later stage, remove) the peptide gate right at the most-sensitive sensing region of a biological nanopore FraC, thus partially blocking its channel and producing a stepwise change in the conductance. Increasing or decreasing the bias while having the peptide gate trapped in the pore stretches or compresses the peptide within the nanopore, thus modulating its conductance in a nonlinear but reproducible manner. We envision a range of applications of this removable-gate nanopore system, e.g. from an element of biological computing circuits to a test bed for probing the elasticity of intrinsically disordered proteins., Accepted Author Manuscript, BN/Chirlmin Joo Lab, BN/Cees Dekker Lab

Proteins are major building blocks of life. The protein content of a cell and an organism provides key information for the understanding of biological processes and disease. Despite the importance of protein analysis, only a handful of techniques are available to determine protein sequences, and these methods face limitations, for example, requiring a sizable amount of sample. Single-molecule techniques would revolutionize proteomics research, providing ultimate sensitivity for the detection of low-abundance proteins and the realization of single-cell proteomics. In recent years, novel single-molecule protein sequencing schemes that use fluorescence, tunnelling currents and nanopores have been proposed. Here, we present a review of these approaches, together with the first experimental efforts towards their realization. We discuss their advantages and drawbacks, and present our perspective on the development of single-molecule protein sequencing techniques., Accepted Author Manuscript, BN/Chirlmin Joo Lab, BN/Cees Dekker Lab

Proteins are major building blocks of life. The protein content of a cell and an organism provides key information for the understanding of biological processes and disease. Despite the importance of protein analysis, only a handful of techniques are available to determine protein sequences, and these methods face limitations, for example, requiring a sizable amount of sample. Single-molecule techniques would revolutionize proteomics research, providing ultimate sensitivity for the detection of low-abundance proteins and the realization of single-cell proteomics. In recent years, novel single-molecule protein sequencing schemes that use fluorescence, tunnelling currents and nanopores have been proposed. Here, we present a review of these approaches, together with the first experimental efforts towards their realization. We discuss their advantages and drawbacks, and present our perspective on the development of single-molecule protein sequencing techniques., Accepted Author Manuscript, BN/Chirlmin Joo Lab, BN/Cees Dekker Lab

RNA interference (RNAi) is an indispensable mechanism for antiviral defense in insects, including mosquitoes that transmit human diseases. To escape this antiviral defense system, viruses encode suppressors of RNAi that prevent elimination of viral RNAs, and thus ensure efficient virus accumulation. Although the first animal Viral Suppressor of RNAi (VSR) was identified more than a decade ago, the molecular basis of RNAi suppression by these viral proteins remains unclear. Here, we developed a single-molecule fluorescence assay to investigate how VSRs inhibit the recognition of viral RNAs by Dcr-2, a key endoribonuclease enzyme in the RNAi pathway. Using VSRs from three insect RNA viruses (Culex Y virus, Drosophila X virus and Drosophila C virus), we reveal bimodal physical interactions between RNA molecules and VSRs. During initial interactions, these VSRs rapidly discriminate short RNA substrates from long dsRNA. VSRs engage nearly irreversible binding with long dsRNAs, thereby shielding it from recognition by Dcr-2. We propose that the length-dependent switch from rapid screening to irreversible binding reflects the main mechanism by which VSRs distinguish viral dsRNA from cellular RNA species such as microRNAs., BN/Chirlmin Joo Lab, Applied Sciences, BN/Technici en Analisten

Using nanopores for single-molecule sequencing of proteins – similar to nanopore-based sequencing of DNA – faces multiple challenges, including unfolding of the complex tertiary structure of the proteins and enforcing their unidirectional translocation through nanopores. Here, we combine molecular dynamics (MD) simulations with single-molecule experiments to investigate the utility of SDS (Sodium Dodecyl Sulfate) to unfold proteins for solid-state nanopore translocation, while simultaneously endowing them with a stronger electrical charge. Our simulations and experiments prove that SDS-treated proteins show a considerable loss of the protein structure during the nanopore translocation. Moreover, SDS-treated proteins translocate through the nanopore in the direction prescribed by the electrophoretic force due to the negative charge impaired by SDS. In summary, our results suggest that SDS causes protein unfolding while facilitating protein translocation in the direction of the electrophoretic force; both characteristics being advantageous for future protein sequencing applications using solid-state nanopores., BN/Chirlmin Joo Lab, BN/Cees Dekker Lab

MicroRNA (miRNA) interferes with the translation of cognate messenger RNA (mRNA) by finding, preferentially binding, and marking it for degradation. To facilitate the search process, Argonaute (Ago) proteins come together with miRNA, forming a dynamic search complex. In this review we use the language of free-energy landscapes to discuss recent single-molecule and high-resolution structural data in the light of theoretical work appropriated from the study of transcription-factor search. We suggest that experimentally observed internal states of the Ago-miRNA search complex may have the explicit biological function of speeding up search while maintaining specificity., BN/Martin Depken Lab, BN/Chirlmin Joo Lab

Using nanopores for single-molecule sequencing of proteins – similar to nanopore-based sequencing of DNA – faces multiple challenges, including unfolding of the complex tertiary structure of the proteins and enforcing their unidirectional translocation through nanopores. Here, we combine molecular dynamics (MD) simulations with single-molecule experiments to investigate the utility of SDS (Sodium Dodecyl Sulfate) to unfold proteins for solid-state nanopore translocation, while simultaneously endowing them with a stronger electrical charge. Our simulations and experiments prove that SDS-treated proteins show a considerable loss of the protein structure during the nanopore translocation. Moreover, SDS-treated proteins translocate through the nanopore in the direction prescribed by the electrophoretic force due to the negative charge impaired by SDS. In summary, our results suggest that SDS causes protein unfolding while facilitating protein translocation in the direction of the electrophoretic force; both characteristics being advantageous for future protein sequencing applications using solid-state nanopores., BN/Chirlmin Joo Lab, BN/Cees Dekker Lab

MicroRNA (miRNA) interferes with the translation of cognate messenger RNA (mRNA) by finding, preferentially binding, and marking it for degradation. To facilitate the search process, Argonaute (Ago) proteins come together with miRNA, forming a dynamic search complex. In this review we use the language of free-energy landscapes to discuss recent single-molecule and high-resolution structural data in the light of theoretical work appropriated from the study of transcription-factor search. We suggest that experimentally observed internal states of the Ago-miRNA search complex may have the explicit biological function of speeding up search while maintaining specificity., BN/Martin Depken Lab, BN/Chirlmin Joo Lab