Single-molecule Techniques Research Articles

Studying chromosome biology with single-molecule resolution in Xenopus laevis egg extracts.

Cell-free extracts from Xenopus laevis eggs are a model system for studying chromosome biology. Xenopus egg extracts can be synchronised in different cell cycle stages, making them useful for studying DNA replication, DNA repair and chromosome organisation. Combining single-molecule approaches with egg extracts is an exciting development being used to reveal molecular mechanisms that are difficult to study using conventional approaches. Fluorescence-based single-molecule imaging of surface-tethered DNAs has been used to visualise labelled protein movements on stretched DNA, the dynamics of DNA–protein complexes and extract-dependent structural rearrangement of stained DNA. Force-based single-molecule techniques are an alternative approach to measure mechanics of DNA and proteins. In this essay, the details of these single-molecule techniques, and the insights into chromosome biology they provide, will be discussed.

Biology on track: single-molecule visualisation of protein dynamics on linear DNA substrates

Single-molecule fluorescence imaging techniques have become important tools in biological research to gain mechanistic insights into cellular processes. These tools provide unique access to the dynamic and stochastic behaviour of biomolecules. Single-molecule tools are ideally suited to study protein-DNA interactions in reactions reconstituted from purified proteins. The use of linear DNA substrates allows for the study of protein-DNA interactions with observation of the movement and behaviour of DNA-translocating proteins over long distances. Single-molecule studies using long linear DNA substrates have revealed unanticipated insights on the dynamics of multi-protein systems. In this review, we provide an overview of recent methodological advances, including the construction of linear DNA substrates. We highlight the versatility of these substrates by describing their application in different single-molecule fluorescence techniques, with a focus on in vitro reconstituted systems. We discuss insights from key experiments on DNA curtains, DNA-based molecular motor proteins, and multi-protein systems acting on DNA that relied on the use of long linear substrates and single-molecule visualisation. The quality and customisability of linear DNA substrates now allows the insertion of modifications, such as nucleosomes, to create conditions mimicking physiologically relevant crowding and complexity. Furthermore, the current technologies will allow future studies on the real-time visualisation of the interfaces between DNA maintenance processes such as replication and transcription.

A High-Performance Fast Capacitance Compensation Method

In the widely used single-molecule Deoxyribonucleic Acid (DNA) sequencing and patch-clamp technique, the fast capacitance (C-Fast) noise in application circuit affects the accuracy of the system measurement. In this paper, a high-performance C-Fast compensation method based on compensation circuit and fuzzy adaptive algorithm is proposed. The model of C-Fast noise is analyzed and a C-Fast compensation circuit is designed, whose parameters can be automatically calculated and adjusted by fuzzy adaptive algorithm. The method is validated through simulations and experiments on the test setup. The results indicate that the proposed method can effectively eliminate the C-Fast noise and improve the measurement accuracy.

Single-molecule micromanipulation studies of methylated DNA

Cytosine methylated at the five-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in longer contour length and increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded “bubbles” of DNA under physiologically relevant stretching forces and supercoiling densities. The flexibility and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.

Single-molecule Electric Switching Induced by Acid-Base Reaction

Electric switches are one of the most fundamental electric circuitries. Recent developments in single-molecule techniques allow us to study various electric-switching phenomena on the single-molecule scale. In this study, the switch of the current through a single molecule of a biphenothiazine derivative was investigated using the break junction technique. The biphenothiazine derivative undergoes acid-base reaction-induced electronic modulation due to the reversible structural transformation between a closed shell and an open shell, resulting in a change in the electronic gap and effective tunneling barrier height. We succeeded in controlling the single-molecule electric conductivity of the biphenothiazine derivative wired to two Au electrodes by allowing the reaction to proceed reversibly on the electrode.

Aggregation-related quenching of LHCII fluorescence in liposomes revealed by single-molecule spectroscopy

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted major light-harvesting complexes of plants (LHCII). By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates bound to the surface. Our results showed that fluorescence lifetime obtained in bulk and in single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII – LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Standard protocol for mass photometry experiments.

Mass photometry (MP) is a relatively new experimental technique with a quickly expanding list of applications. Using optical detection, MP measures the mass of individual molecules to obtain molecular mass distributions of proteins and other biomolecules in solution. The combination of speed, sensitivity, and very low sample consumption with label- and immobilization-free detection sets MP apart from other analytical methods. An increasing number of laboratories incorporates mass photometry as a routine sample analysis technique. However, MP measurements can sometimes be challenging, especially for users without previous experience with single-molecule techniques. Here, we present a protocol for the determination of protein molecular mass distributions by MP. It describes the sample and materials preparation as well as data collection and analysis. The advantages and limitations of this technique and the potential sources of artifacts are also given. This protocol can be used by new MP users and serve as a checklist for laboratories routinely performing MP experiments to guide consistent data collection and documentation.

Topographically smooth and stable supported lipid bilayer for high-resolution AFM studies.

The cellular membrane has been identified to play a critical role in various biological processes including the assembly of biological systems. Membranes are complex, primarily two-dimensional assemblies with varied lipid compositions depending on the particular region of the cell. Supported lipid bilayers are considered as appropriate models for physio-chemical studies of membranes including numerous single molecule techniques. Atomic force microscopy (AFM) as a topographic technique is a fully appropriate single molecule technique capable of direct observation of molecular processes on membranes. However, reliable experimental AFM studies require the preparation of the bilayer with a sub-nanometer smooth morphology, which remains stable over long-time observation. Here we present the methodology, which allows one to prepare a smooth, stable, structurally homogeneous lipid bilayer without the presence of any trapped vesicles. We described the application of such lipid bilayers to probe time-dependent early stages of aggregation of monomeric amyloid proteins. Importantly, the proposed methodology can be extended to bilayers with various compositions, by incorporating different lipids for on-membrane aggregation study including cholesterol. Furthermore, this methodology development allowed us to monitor the aggregation of amyloid protein at its physiologically relevant low protein concentration. The flexibility of altering the membrane composition allows to identify the specific role of a particular lipid towards the aggregation kinetics, revealing the plausible mechanism of disease development.

Carbon Dot Blinking Enables Accurate Molecular Counting at Nanoscale Resolution.

Accurate counting of single molecules at nanoscale resolution is essential for the study of molecular interactions and distribution in subcellular fractions. By using small-sized carbon dots (CDs), we have now developed a quantitative single-molecule localization microscopy technique (qSMLM) based on spontaneous blinking to count single molecules with a localization precision of 10 nm, which can be accomplished on conventional microscopes without sophisticated laser control. We explore and adapt the blinking of CDs with diverse structures and demonstrate a counting accuracy of >97% at a molecular density of 500 per μm2. When applied to G-protein coupled receptors on a cell membrane, we discriminated receptor oligomerization and clustering and revealed ligand-regulated receptor distribution patterns. This is the first example of adapting nanoparticle self-blinking for molecular counting, and this demonstrates the power of CDs as SMLM probes to reliably decipher sub-diffraction structures that mediate crucial biological functions.

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis.

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry.

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

Spectral Clustering to Analyze the Hidden Events in Single-Molecule Break Junctions

The single-molecule break junction technique provides a high-throughput method to explore the charge transport phenomena through a molecular junction at the ultimate scale of a single molecule. The...

Statistical and Biophysical Analysis of Force-Pulling Optical Tweezers Data in Complex Protein Systems

Single molecule techniques such as optical tweezers represent a powerful collection of emerging methods in biophysics, but their new approach to biological systems brings new challenges in extracting meaningful results from the data they produce. By working to account for the stochasticity that arises from studying the behavior of proteins one molecule at a time and considering how the system responds to force, new insights can be gained from optical tweezers data. We show that model-based clustering along with bootstrapping successfully identifies and characterizes five states along the cofactor-binding coupled folding pathway of the Drosophila cryptochrome, which allowed for the extraction of all of the kinetic rate constants connecting these states.

Single-Molecule Imaging Reveals the Mechanism Underlying Histone Loading of Schizosaccharomyces pombe AAA+ ATPase Abo1.

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1-DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.

Shining Light on the ‘Dark Matter’ of the Nuclear Pore Complex

The nuclear pore complex (NPC) is the gatekeeper for the nucleocytoplasmic transport of macromolecules in eukaryotic cells. The centralchannel of the NPC is filled with intrinsically disordered proteins (IDPs) enriched in phenylalanine glycine (FG) residues, known as FG-Nups. Due to current technical limitations, how the FG-Nups line the NPC channel and form a selective permeability barrier for transport, still remains elusive. Here we develop a novel high-resolution imaging tool that allows direct visualization of FG-Nups in situ. We generate artificial organelles in the cell that allow to exclusively expand the genetic code for targeted FG-Nups. We use them to incorporate non-canonical amino acids into FG-Nups with single-residue precision, which we subsequently label with small, photostable fluorescent dyes via click-chemistry. This labeling method ensures minimal interference with the structures and biological activities of the labeled FG-Nups. By synergistically combining this labeling method with a double strategy for imaging (i.e. super-resolution microscopy and fluorescence lifetime imaging microscopy), we can bridge the resolution boundary between single-molecule and super-resolution techniques, and acquire complementary spatial information about FG-Nups in situ. This work paves the way for better understanding the conformations, dynamics, and functions of FG-Nups in the cell. The developed novel tool is generally applicable to study the plasticity and functions of other IDPs, filling the current technology gap in the field.

Single-Molecule Insights into Allosteric Regulation of the Nuclear Receptor Protein RXRα

Nuclear receptors are crucial transcription factors that respond to a broad range of external signaling ligands by binding cognate DNA response elements and switching on transcription. As such, these proteins form tightly regulated, multi-component allosteric systems. RXRα is one of the key players in this protein family for its role as a promiscuous heterodimerization partner. We employ single-molecule fluorescence techniques to investigate the conformational dynamics of RXRα's ligand binding domain (LBD)—the essential regulatory domain. It has been proposed that ligand binding changes the conformation of helix 12 of the LBD and positions it in a conformation favoring coactivator peptide binding. Our single-molecule FRET studies, however, demonstrate that helix 12 undergoes a substantial conformational change only after the coactivator peptide binds. This suggests an unexpected allosteric mechanism of nuclear receptor activation, which we investigate. In particular, we address the questions: on what time scales do the conformational changes of helix 12 take place? and how are its dynamics affected by modulations in other parts of RXRα and in its binding partners? Our studies will pave the way to better understanding of the complex regulatory machinery embodied in nuclear receptor function.

Single-Chain Dimensions of an Archetypal Phase-Separating Intrinsically Disordered Protein Region

Membrane-less organelles (MLOs) are non-stoichiometric assemblies of concentrated biomolecules lacking a surrounding lipid membrane and form via liquid-liquid phase separation (LLPS). They facilitate a variety of cellular processes, and the dysregulation of phase separation can lead to debilitating diseases. Intrinsically disordered regions (IDRs) in proteins have often been found to drive phase separation. Due to lack of structure, disordered proteins are challenging to study with traditional structural biology methods, and this combined with crowded dense phase environments requires careful experimental design in order to extract quantitative information. We are directing our studies at an archetypal IDR that undergoes homotypic phase separation, i.e. the prion-like domain (PLD) of hnRNPA1. Here we use single-molecule fluorescence techniques to explore the dimensions and dynamics of hnRNPA1 in light and dense phases. We characterize single-chain behavior in response to salt concentration, which is used to induce phase separation, and elucidate how this is modulated by sequence variation. Initial measurements show that the PLD is a compact disordered protein in the dilute regime and that modulation of the driving force for phase separation also modulates the single-chain dimensions. This work will provide insights into structural and dynamic features of phase-separating biomolecules and will enable the investigation of maturation effects responsible for disease states.

Reconstructing Protein Structure and Dynamics from Experimental Observables

Single-molecule experimental techniques track the real-time dynamics of molecules by recording a small number of experimental observables. Following these observables provides a coarse-grained representation of the conformational dynamics but does not furnish an atomistic representation of the instantaneous molecular structure. Remarkably, a theorem from dynamical systems asserts that these time series can, under quite general conditions, contain sufficient information to reconstruct the atomistic structure of the molecule.

Combining Fluorescence Microscopy and Acoustic Force Spectroscopy to Measure DNA and Protein Mechanics

Acoustic Force Spectroscopy (AFS) is a single-molecule force spectroscopy technique that uses standing ultrasound waves generated by a piezo element to apply forces to DNA, proteins and cells in a well-controlled and multiplexed way. Here, we integrate AFS with fluorescence microscopy to allow correlations between force and fluorescence signatures. This is particularly useful to define the number of (fluorescently labelled) ligands bound to a DNA or protein molecule, allowing us to extract multiple populations from the same measurement.

MYC Amplifies Gene Expression Through Global Changes in Transcription Factor Dynamics

The MYC oncogene has been studied intensively for decades, yet there is still intense debate over how this transcription factor controls gene expression. Here, we seek to answer these questions with an in vivo readout of the role of MYC in discrete events of gene expression in single cells. We engineered an optogenetic variant of MYC (Pi-MYC) and combined this tool with single-molecule RNA and protein imaging techniques to investigate the role of MYC in modulating transcriptional bursts and transcription factor binding dynamics in human cells. We find that the immediate consequence of MYC overexpression is to increase the duration rather than frequency of bursts, a role that appears unique among transcription factors. We further propose that the mechanism by which MYC exerts global effects on the active period of genes is by altering the binding dynamics of transcription factors involved in RNA Polymerase II complex assembly and productive elongation.