Single-molecule Methods Research Articles

Rapid extraction and kinetic analysis of protein complexes from single cells

Proteins contribute to cell biology by forming dynamic, regulated interactions, and measuring these interactions is a foundational approach in biochemistry. We present a rapid, quantitative in vivo assay for protein-protein interactions, based on optical cell lysis followed by time-resolved single-molecule analysis of protein complex binding to an antibody-coated substrate. We show that our approach has better reproducibility, higher dynamic range, and lower background than previous single-molecule pull-down assays. Furthermore, we demonstrate that by monitoring cellular protein complexes over time after cell lysis, we can measure the dissociation rate constant of a cellular protein complex, providing information about binding affinity and kinetics. Our dynamic single-cell, single-molecule pull-down method thus approaches the biochemical precision that is often sought from in vitro assays while being applicable to native protein complexes isolated from single cells in vivo.

In silico assessment of a novel single-molecule protein fingerprinting method employing fragmentation and nanopore detection

SummaryThe identification of proteins at the single-molecule level would open exciting new venues in biological research and disease diagnostics. Previously, we proposed a nanopore-based method for protein identification called chop-n-drop fingerprinting, in which the fragmentation pattern induced and measured by a proteasome-nanopore construct is used to identify single proteins. In the simulation study presented here, we show that 97.1% of human proteome constituents are uniquely identified under close to ideal measuring circumstances, using a simple alignment-based classification method. We show that our method is robust against experimental error, as 69.4% can still be identified if the resolution is twice as low as currently attainable, and 10% of proteasome restriction sites and protein fragments are randomly ignored. Based on these results and our experimental proof of concept, we argue that chop-n-drop fingerprinting has the potential to make cost-effective single-molecule protein identification feasible in the near future.

The MAVS Immune Recognition Pathway in Viral Infection and Sepsis.

Significance: It is estimated that close to 50 million cases of sepsis result in over 11 million annual fatalities worldwide. The pathognomonic feature of sepsis is a dysregulated inflammatory response arising from viral, bacterial, or fungal infections. Immune recognition of pathogen-associated molecular patterns is a hallmark of the host immune defense to combat microbes and to prevent the progression to sepsis. Mitochondrial antiviral signaling protein (MAVS) is a ubiquitous adaptor protein located at the outer mitochondrial membrane, which is activated by the cytosolic pattern recognition receptors, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation associated gene 5 (MDA5), following binding of viral RNA agonists. Recent Advances: Substantial progress has been made in deciphering the activation of the MAVS pathway with its interacting proteins, downstream signaling events (interferon [IFN] regulatory factors, nuclear factor kappa B), and context-dependent type I/III IFN response. Critical Issues: In the evolutionary race between pathogens and the host, viruses have developed immune evasion strategies for cleavage, degradation, or blockade of proteins in the MAVS pathway. For example, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M protein and ORF9b protein antagonize MAVS signaling and a protective type I IFN response. Future Directions: The role of MAVS as a sensor for nonviral pathogens, host cell injury, and metabolic perturbations awaits better characterization in the future. New technical advances in multidimensional single-cell analysis and single-molecule methods will accelerate the rate of new discoveries. The ultimate goal is to manipulate MAVS activities in the form of immune-modulatory therapies to combat infections and sepsis. Antioxid. Redox Signal. 35, 1376-1392.

Single-Molecule Methods for Investigating the Double-Stranded DNA Bendability.

The various DNA-protein interactions associated with the expression of genetic information involve double-stranded DNA (dsDNA) bending. Due to the importance of the formation of the dsDNA bending structure, dsDNA bending properties have long been investigated in the biophysics field. Conventionally, DNA bendability is characterized by innate averaging data from bulk experiments. The advent of single-molecule methods, such as atomic force microscopy, optical and magnetic tweezers, tethered particle motion, and single-molecule fluorescence resonance energy transfer measurement, has provided valuable tools to investigate not only the static structures but also the dynamic properties of bent dsDNA. Here, we reviewed the single-molecule methods that have been used for investigating dsDNA bendability and new findings related to dsDNA bending. Single-molecule approaches are promising tools for revealing the unknown properties of dsDNA related to its bending, particularly in cells.

Correlative approaches in single-molecule biophysics: A review of the progress in methods and applications

Here, we discuss a collection of cutting-edge techniques and applications in use today by some of the leading experts in the field of correlative approaches in single-molecule biophysics. A key difference in emphasis, compared with traditional single-molecule biophysics approaches detailed previously, is on the emphasis of the development and use of complex methods which explicitly combine multiple approaches to increase biological insights at the single-molecule level. These so-called correlative single-molecule biophysics methods rely on multiple, orthogonal tools and analysis, as opposed to any one single driving technique. Importantly, they span both in vivo and in vitro biological systems as well as the interfaces between theory and experiment in often highly integrated ways, very different to earlier traditional non-integrative approaches. The first applications of correlative single-molecule methods involved adaption of a range of different experimental technologies to the same biological sample whose measurements were synchronised. However, now we find a greater flora of integrated methods emerging that include approaches applied to different samples at different times and yet still permit useful molecular-scale correlations to be performed. The resultant findings often enable far greater precision of length and time scales of measurements, and a more nuanced understanding of the interplay between different processes in the same cell. Many new correlative single-molecule biophysics techniques also include more complex, physiologically relevant approaches as well as an increasing number that combine of approaches advanced computational methods and mathematical analysis with experimental tools. Here, we review the motivation behind the development of correlative single-molecule microscopy methods, its history and recent progress in the field.

Single-Molecule Insights Into the Dynamics of Replicative Helicases.

Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork. The replicative helicases of the model systems bacteriophages T4 and T7, Escherichia coli and Saccharomyces cerevisiae have been extensively studied and characterized using biochemical methods. While powerful, their averaging over ensembles of molecules and reactions makes it challenging to uncover information related to intermediate states in the unwinding process and the dynamic helicase interactions within the replisome. Here, we describe single-molecule methods that have been developed in the last few decades and discuss the new details that these methods have revealed about replicative helicases. Applying methods such as FRET and optical and magnetic tweezers to individual helicases have made it possible to access the mechanistic aspects of unwinding. It is from these methods that we understand that the replicative helicases studied so far actively translocate and then passively unwind DNA, and that these hexameric enzymes must efficiently coordinate the stepping action of their subunits to achieve unwinding, where the size of each step is prone to variation. Single-molecule fluorescence microscopy methods have made it possible to visualize replicative helicases acting at replication forks and quantify their dynamics using multi-color colocalization, FRAP and FLIP. These fluorescence methods have made it possible to visualize helicases in replication initiation and dissect this intricate protein-assembly process. In a similar manner, single-molecule visualization of fluorescent replicative helicases acting in replication identified that, in contrast to the replicative polymerases, the helicase does not exchange. Instead, the replicative helicase acts as the stable component that serves to anchor the other replication factors to the replisome.

Imaging protein aggregates in the serum and cerebrospinal fluid in Parkinson's disease.

Aggregation of α-synuclein plays a key role in the development of Parkinson’s disease. Soluble aggregates are present not only within human brain but also the CSF and blood. Characterizing the aggregates present in these biofluids may provide insights into disease mechanisms and also have potential for aiding diagnosis.We used two optical single-molecule imaging methods called aptamer DNA-PAINT and single-aggregate confocal fluorescence, together with high-resolution atomic force microscopy for specific detection and characterization of individual aggregates with intermolecular β-sheet structure, present in the CSF and serum of 15 early stage Parkinson’s disease patients compared to 10 healthy age-matched controls.We found aggregates ranging in size from 20 nm to 200 nm, in both CSF and serum. There was a difference in aggregate size distribution between Parkinson’s disease and control groups with a significantly increased number of larger aggregates (longer than 150 nm) in the serum of patients with Parkinson’s disease. To determine the chemical composition of the aggregates, we performed aptamer DNA-PAINT on serum following α-synuclein and amyloid-β immunodepletion in an independent cohort of 11 patients with early stage Parkinson’s disease and 10 control subjects. β-Sheet aggregates in the serum of Parkinson’s disease patients were found to consist of, on average, 50% α-synuclein and 50% amyloid-β in contrast to 30% α-synuclein and 70% amyloid-β in control serum [the differences in the proportion of these aggregates were statistically significant between diseased and control groups (P = 1.7 × 10−5 for each species)]. The ratio of the number of β-sheet α-synuclein aggregates to β-sheet amyloid-β aggregates in serum extracted using our super-resolution method discriminated Parkinson’s disease cases from controls with an accuracy of 98.2% (AUC = 98.2%, P = 4.3 × 10−5).Our data suggest that studying the protein aggregates present in serum can provide information about the disruption of protein homeostasis occurring in Parkinson’s disease and warrants further investigation as a potential biomarker of disease.

Protein detection in blood with single-molecule imaging.

The ability to characterize individual biomarker protein molecules in patient blood samples could enable diagnosis of diseases at an earlier stage, when treatment is typically more effective. Single-molecule imaging offers a promising approach to accomplish this goal. However, thus far, single-molecule imaging methods have not been translated into the clinical setting. The detection limit of these methods has been confined to the picomolar (10-12 M) range, several orders of magnitude higher than the circulating concentrations of biomarker proteins present in many diseases. Here, we describe single-molecule augmented capture (SMAC), a single-molecule imaging technique to quantify and characterize individual protein molecules of interest down to the subfemtomolar (<10-15 M) range. We demonstrate SMAC in a variety of applications with human blood samples, including the analysis of disease-associated secreted proteins, membrane proteins, and rare intracellular proteins. SMAC opens the door to the application of single-molecule imaging in noninvasive disease profiling.

Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization.

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that plays important roles in DNA repair, chromatin organization and transcription regulation. Although binding and activation of PARP1 by DNA damage sites has been extensively studied, little is known about how PARP1 binds to long stretches of undamaged DNA and how it could shape chromatin architecture. Here, using single-molecule techniques, we show that PARP1 binds and condenses undamaged, kilobase-length DNA subject to sub-piconewton mechanical forces. Stepwise decondensation at high force and DNA braiding experiments show that the condensation activity is due to the stabilization of DNA loops by PARP1. PARP inhibitors do not affect the level of condensation of undamaged DNA but act to block condensation reversal for damaged DNA in the presence of NAD+ Our findings suggest a mechanism for PARP1 in the organization of chromatin structure.

Direct imaging of single-molecule electrochemical reactions in solution.

Chemical reactions tend to be conceptualized in terms of individual molecules transforming into products, but are usually observed in experiments that probe the average behaviour of the ensemble. Single-molecule methods move beyond ensemble averages and reveal the statistical distribution of reaction positions, pathways and dynamics1-3. This has been shown with optical traps and scanning probe microscopy manipulating and observing individual reactions at defined locations with high spatial resolution4,5, and with modern optical methods using ultrasensitive photodetectors3,6,7 that enable high-throughput single-molecule measurements. However, effective probing of single-molecule solution chemistry remains challenging. Here we demonstrate optical imaging of single-molecule electrochemical reactions7 in aqueous solution and its use for super-resolution microscopy. The method utilizes a chemiluminescent reaction involving a ruthenium complex electrochemically generated at an electrode8, which ensures minimal background signal. This allows us to directly capture single photons of the electrochemiluminescence of individual reactions, and to develop super-resolved electrochemiluminescence microscopy for imaging the adhesion dynamics of live cells with high spatiotemporal resolution. We anticipate that our method will advance the fundamental understanding of electrochemical reactions and prove useful for bioassays and cell-imaging applications.

Single-molecule fluorescence vistas of how lipids regulate membrane proteins.

The study of membrane proteins is undergoing a golden era, and we are gaining unprecedented knowledge on how this key group of proteins works. However, we still have only a basic understanding of how the chemical composition and the physical properties of lipid bilayers control the activity of membrane proteins. Single-molecule (SM) fluorescence methods can resolve sample heterogeneity, allowing to discriminate between the different molecular populations that biological systems often adopt. This short review highlights relevant examples of how SM fluorescence methodologies can illuminate the different ways in which lipids regulate the activity of membrane proteins. These studies are not limited to lipid molecules acting as ligands, but also consider how the physical properties of the bilayer can be determining factors on how membrane proteins function.

Time-Course Transcriptome Profiling of a Poxvirus Using Long-Read Full-Length Assay.

Viral transcriptomes that are determined using first- and second-generation sequencing techniques are incomplete. Due to the short read length, these methods are inefficient or fail to distinguish between transcript isoforms, polycistronic RNAs, and transcriptional overlaps and readthroughs. Additionally, these approaches are insensitive for the identification of splice and transcriptional start sites (TSSs) and, in most cases, transcriptional end sites (TESs), especially in transcript isoforms with varying transcript ends, and in multi-spliced transcripts. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome has a high diversity of TSSs and TESs, and a high degree of polycistronism that leads to enormous complexity. We applied single-molecule, real-time, and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of VACV gene expression.

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay

Structural basis of early translocation events on the ribosome

Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2–20 per second) and with a low error rate (around 10−3 to 10−5 at each step) over thousands of cycles1. The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome. Our findings reveal that the bacterial GTPase elongation factor G specifically engages spontaneously achieved ribosome conformations while in an active, GTP-bound conformation to unlock and initiate peptidyl-tRNA translocation. These findings suggest that processes intrinsic to the pre-translocation ribosome complex can regulate the rate of protein synthesis, and that energy expenditure is used later in the translocation mechanism than previously proposed.

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

Single-Molecule Techniques to Study Chromatin.

Besides the basic organization in nucleosome core particles (NCPs), eukaryotic chromatin is further packed through interactions with numerous protein complexes including transcription factors, chromatin remodeling and modifying enzymes. This nucleoprotein complex provides the template for many important biological processes, such as DNA replication, transcription, and DNA repair. Thus, to understand the molecular basis of these DNA transactions, it is critical to define individual changes of the chromatin structure at precise genomic regions where these machineries assemble and drive biological reactions. Single-molecule approaches provide the only possible solution to overcome the heterogenous nature of chromatin and monitor the behavior of individual chromatin transactions in real-time. In this review, we will give an overview of currently available single-molecule methods to obtain mechanistic insights into nucleosome positioning, histone modifications and DNA replication and transcription analysis—previously unattainable with population-based assays.

Deep-Learning-Assisted Single-Molecule Tracking on a Live Cell Membrane.

Single-molecule fluorescence imaging is a powerful tool to study protein function by tracking molecular position and distribution, but the precise and rapid identification of dynamic molecules remains challenging due to the heterogeneous distribution and interaction of proteins on the live cell membrane. We now develop a deep-learning (DL)-assisted single-molecule imaging method that can precisely distinguish the monomer and complex for rapid and real-time tracking of protein interaction. This DL-based model, which comprises convolutional layers, max pooling layers, and fully connected layers, is trained to reach an accuracy of >98% for identifying monomer and complex. We use this method to investigate the dynamic process of chemokine receptor CXCR4 on the live cell membrane during the early signaling stage. The results show that, upon ligand activation, the CXCR4 undergoes a dynamic process of forming a receptor complex. We further demonstrate that the CXCR4 complex tends to be internalized at 2.5-fold higher rate into the cell interior than the monomer via the clathrin-dependent pathway. This study is the first example to scrutinize the early signaling process of CXCR4 at the single-molecule level on the live cell membrane. We envision that this DL-assisted imaging method would be a broadly useful technique to study more protein families for elucidating their physiological and pathological functions.

Perspectives and Benefits of High-Throughput Long-Read Sequencing in Microbial Ecology.

Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example, by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities, such as rapid molecular diagnostics and direct RNA sequencing, and both Pacific Biosciences (PacBio) and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long-read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.

DNAscent v2: detecting replication forks in nanopore sequencing data with deep learning

BackgroundMeasuring DNA replication dynamics with high throughput and single-molecule resolution is critical for understanding both the basic biology behind how cells replicate their DNA and how DNA replication can be used as a therapeutic target for diseases like cancer. In recent years, the detection of base analogues in Oxford Nanopore Technologies (ONT) sequencing reads has become a promising new method to supersede existing single-molecule methods such as DNA fibre analysis: ONT sequencing yields long reads with high throughput, and sequenced molecules can be mapped to the genome using standard sequence alignment software.ResultsThis paper introduces DNAscent v2, software that uses a residual neural network to achieve fast, accurate detection of the thymidine analogue BrdU with single-nucleotide resolution. DNAscent v2 also comes equipped with an autoencoder that interprets the pattern of BrdU incorporation on each ONT-sequenced molecule into replication fork direction to call the location of replication origins termination sites. DNAscent v2 surpasses previous versions of DNAscent in BrdU calling accuracy, origin calling accuracy, speed, and versatility across different experimental protocols. Unlike NanoMod, DNAscent v2 positively identifies BrdU without the need for sequencing unmodified DNA. Unlike RepNano, DNAscent v2 calls BrdU with single-nucleotide resolution and detects more origins than RepNano from the same sequencing data. DNAscent v2 is open-source and available at https://github.com/MBoemo/DNAscent.ConclusionsThis paper shows that DNAscent v2 is the new state-of-the-art in the high-throughput, single-molecule detection of replication fork dynamics. These improvements in DNAscent v2 mark an important step towards measuring DNA replication dynamics in large genomes with single-molecule resolution. Looking forward, the increase in accuracy in single-nucleotide resolution BrdU calls will also allow DNAscent v2 to branch out into other areas of genome stability research, particularly the detection of DNA repair.