Single-molecule Techniques Research Articles

100th Anniversary of Macromolecular Science Viewpoint: Enabling Advances in Fluorescence Microscopy Techniques.

In the past few decades there has been a revolution in the field of optical microscopy with emerging capabilities such as super-resolution and single-molecule fluorescence techniques. Combined with the classical advantages of fluorescence imaging, such as chemical labeling specificity, and noninvasive sample preparation and imaging, these methods have enabled significant advances in our polymer community. This Viewpoint discusses several of these capabilities and how they can uniquely offer information where other characterization techniques are limited. Several examples are highlighted that demonstrate the ability of fluorescence microscopy to understand key questions in polymer science such as single-molecule diffusion and orientation, 3D nanostructural morphology, and interfacial and multicomponent dynamics. Finally, we briefly discuss opportunities for further advances in techniques that may allow them to make an even greater contribution in polymer science.

The dynamic landscape of transcription initiation in yeast mitochondria

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.

Rate-dependent force–extension models for single-molecule force spectroscopy experiments

Single-molecule force spectroscopy techniques allow for the measurement of several static and dynamic features of macromolecules of biological origin. In particular, atomic force microscopy, used with a variable pulling rate, provides valuable information on the folding/unfolding dynamics of proteins. We propose here two different models able to describe the out-of-equilibrium statistical mechanics of a chain composed of bistable units. These latter represent the protein domains, which can be either folded or unfolded. Both models are based on the Langevin approach and their implementation allows for investigating the effect of the pulling rate and of the device intrinsic elasticity on the chain unfolding response. The theoretical results (both analytical and numerical) have been compared with experimental data concerning the unfolding of the titin and filamin proteins, eventually obtaining a good agreement over a large range of the pulling rates.

Quantum Yield Limits for the Detection of Single-Molecule Fluorescence Enhancement by a Gold Nanorod

Fluorescence-based single-molecule optical detection techniques are widely chosen over other methods, owing to the ease of background screening and better signal-to-noise throughput. Nonetheless, the methodology still suffers from limitations imposed by weak emitting properties of most molecules. Plasmonic nanostructures, such as gold nanorods, can significantly enhance the fluorescence signal of a weak emitter, extending the application of these techniques to a wider range of species. In this work, we explore the lower limit of fluorescence quantum yield for single-molecule detection, using a single gold nanorod to enhance molecular fluorescence. We specifically designed an infrared dye with the extremely low quantum yield of 10–4 and a comparatively large Stokes shift of 3000 cm–1 to demonstrate single-molecule detection by fluorescence enhancement. This example allows us to discuss more general cases. We estimate theoretically the optimal excitation wavelength and the plasmon resonance of the rod that maximize the fluorescence signals. We then confirm experimentally the detection of single-molecule fluorescence with an enhancement factor of 3 orders of magnitude for the quantum yield 10–4. Theoretical simulations indicate that single-molecule signals should be detectable for molecules with quantum yield as low as 10–6, provided the dwell time of the molecules in the plasmonic hot spot is long enough.

Sensing with Nanopores and Aptamers: A Way Forward.

In the 90s, the development of a novel single molecule technique based on nanopore sensing emerged. Preliminary improvements were based on the molecular or biological engineering of protein nanopores along with the use of nanotechnologies developed in the context of microelectronics. Since the last decade, the convergence between those two worlds has allowed for biomimetic approaches. In this respect, the combination of nanopores with aptamers, single-stranded oligonucleotides specifically selected towards molecular or cellular targets from an in vitro method, gained a lot of interest with potential applications for the single molecule detection and recognition in various domains like health, environment or security. The recent developments performed by combining nanopores and aptamers are highlighted in this review and some perspectives are drawn.

Mismatch sensing by nucleofilament deciphers mechanism of RecA-mediated homologous recombination

Recombinases polymerize along single-stranded DNA (ssDNA) at the end of a broken DNA to form a helical nucleofilament with a periodicity of ∼18 bases. The filament catalyzes the search and checking for homologous sequences and promotes strand exchange with a donor duplex during homologous recombination (HR), the mechanism of which has remained mysterious since its discovery. Here, by inserting mismatched segments into donor duplexes and using single-molecule techniques to catch transient intermediates in HR, we found that, even though 3 base pairs (bp) is still the basic unit, both the homology checking and the strand exchange may proceed in multiple steps at a time, resulting in ∼9-bp large steps on average. More interestingly, the strand exchange is blocked remotely by the mismatched segment, terminating at positions ∼9 bp before the match-mismatch joint. The homology checking and the strand exchange are thus separated in space, with the strand exchange lagging behind. Our data suggest that the strand exchange progresses like a traveling wave in which the donor DNA is incorporated successively into the ssDNA-RecA filament to check homology in ∼9-bp steps in the frontier, followed by a hypothetical transitional segment and then the post-strand-exchanged duplex.

Hitchhiker model for Laplace diffusion processes.

Brownian motion is a Gaussian process describing normal diffusion with a variance increasing linearly with time. Recently, intracellular single-molecule tracking experiments have recorded exponentially decaying propagators, a phenomenon called Laplace diffusion. Inspired by these developments we study a many-body approach, called the Hitchhiker model, providing a microscopic description of the widely observed behavior. Our model explains how Laplace diffusion is controlled by size fluctuations of single molecules, independently of the diffusion law which they follow. By means of numerical simulations Laplace diffusion is recovered and we show how single-molecule tracking and data analysis, in a many-body system, is highly nontrivial as tracking of a single particle or many in parallel yields vastly different estimates for the diffusivity. We quantify the differences between these two commonly used approaches, showing how the single-molecule estimate of diffusivity is larger if compared to the full tagging method.

Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

As the basic building blocks of chromatin, nucleosomes play a key role in dictating the accessibility of the eukaryotic genome. Consequently, nucleosomes are involved in essential genomic transactions such as DNA transcription, replication and repair. In order to unravel the mechanisms by which nucleosomes can influence, or be altered by, DNA-binding proteins, single-molecule techniques are increasingly employed. To this end, DNA molecules containing a defined series of nucleosome positioning sequences are often used to reconstitute arrays of nucleosomes in vitro. Here, we describe a novel method to prepare DNA molecules containing defined arrays of the ‘601’ nucleosome positioning sequence by exploiting Gibson Assembly cloning. The approaches presented here provide a more accessible and efficient means to generate arrays of nucleosome positioning motifs, and facilitate a high degree of control over the linker sequences between these motifs. Nucleosomes reconstituted on such arrays are ideal for interrogation with single-molecule techniques. To demonstrate this, we use dual-trap optical tweezers, in combination with fluorescence microscopy, to monitor nucleosome unwrapping and histone localisation as a function of tension. We reveal that, although nucleosomes unwrap at ~20 pN, histones (at least histone H3) remain bound to the DNA, even at tensions beyond 60 pN.

Methods for spatial and temporal imaging of the different steps involved in RNA processing at single-molecule resolution.

RNA plays a quintessential role as a messenger of information from genotype (DNA) to phenotype (proteins), as well as acts as a regulatory molecule (noncoding RNAs). All steps in the journey of RNA from synthesis (transcription), splicing, transport, localization, translation, to its eventual degradation, comprise important steps in gene expression, thereby controlling the fate of the cell. This lifecycle refers to the majority of RNAs (primarily mRNAs), but not other RNAs such as tRNAs. Imaging these processes in fixed cells and in live cells has been an important tool in developing an understanding of the regulatory steps in RNAs journey. Single-cell and single-molecule imaging techniques enable a much deeper understanding of cellular biology, which is not possible with bulk studies involving RNA isolated from a large pool of cells. Classic techniques, such as fluorescence in situ hybridization (FISH), as well as more recent aptamer-based approaches, have provided detailed insights into RNA localization, and have helped to predict the functions carried out by many RNA species. However, there are still certain processing steps that await high-resolution imaging, which is an exciting and upcoming area of research. In this review, we will discuss the methods that have revolutionized single-molecule resolution imaging in general, the steps of RNA processing in which these methods have been used, and new emerging technologies. This article is categorized under: RNA Export and Localization > RNA Localization RNA Methods > RNA Analyses in Cells RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions.

Constructing Dual-Molecule Junctions to Probe Intermolecular Crosstalk.

Understanding and controlling charge transport across multiple parallel molecules are fundamental to the creation of innovative functional electronic components, as future molecular devices will likely be multimolecular. The smallest possible molecular ensemble to address this challenge is a dual-molecule junction device, which has potential to unravel the effects of intermolecular crosstalk on electronic transport at the molecular level that cannot be elucidated using either conventional single-molecule or self-assembled monolayer (SAM) techniques. Herein, we demonstrate the fabrication of a scanning tunneling microscopy (STM) dual-molecule junction device, which utilizes noncovalent interactions and allows for direct comparison to the conventional STM single-molecule device. STM-break junction (BJ) measurements reveal a decrease in conductance of 10% per molecule from the dual-molecule to the single-molecule junction device. Quantum transport simulations indicate that this decrease is attributable to intermolecular crosstalk (i.e., intermolecular π-π interactions), with possible contributions from substrate-mediated coupling (i.e., molecule-electrode). This study provides the first experimental evidence to interpret intermolecular crosstalk in electronic transport at the STM-BJ level and translates the experimental observations into meaningful molecular information to enhance our fundamental knowledge of this subject matter. This approach is pertinent to the design and development of future multimolecular electronic components and also to other dual-molecular systems where such crosstalk is mediated by various noncovalent intermolecular interactions (e.g., electrostatic and hydrogen bonding).

Direct observation of water-mediated single-proton transport between hBN surface defects.

Aqueous proton transport at interfaces is ubiquitous and crucial for a number of fields, ranging from cellular transport and signalling, to catalysis and membrane science. However, due to their light mass, small size and high chemical reactivity, uncovering the surface transport of single protons at room temperature and in an aqueous environment has so far remained out-of-reach of conventional atomic-scale surface science techniques, such as scanning tunnelling microscopy. Here, we use single-molecule localization microscopy to resolve optically the transport of individual excess protons at the interface of hexagonal boron nitride crystals and aqueous solutions at room temperature. Single excess proton trajectories are revealed by the successive protonation and activation of optically active defects at the surface of the crystal. Our observations demonstrate, at the single-molecule scale, that the solid/water interface provides a preferential pathway for lateral proton transport, with broad implications for molecular charge transport at liquid interfaces.

Single-Molecule Sensing of DNA Intercalating Drugs in Water.

The occurrence of pharmaceutical residues in surface water is raising environmental concern. To accompany the evolution of measures for natural resources protection, sensing methods enabling sensitive and rapid water quality monitoring are needed. We recently managed the parallelization of the Tethered Particle Motion (TPM), a single molecule technique, sensitive to the conformational changes of DNA. Here, we investigate the capacity of high throughput TPM (htTPM) to detect drugs that intercalate into DNA. As a proof-of-concept we analyze the htTPM signal for two DNA intercalating dyes, namely, YOYO-1 and SYTOX orange. The efficient detection of intercalating drugs is then demonstrated with doxorubicin. We further evaluate the possibility to detect carbamazepine, an antiepileptic massively prescribed and persistent in water, which had been described to interact with DNA through intercalation. Our results corroborated by other techniques show that, in fact, carbamazepine is not a DNA intercalator. The comparison of the results obtained with different aqueous buffers and solutions allows us to identify optimal conditions for the monitoring of intercalation compounds by htTPM.

Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics.

The field of proteomics has expanded recently with more sensitive techniques for the bulk measurement of peptides as well as single-molecule techniques. One limiting factor for some of these methods is the need for multiple chemical derivatizations and highly pure proteins free of contaminants. We demonstrate a solid-phase capture-release strategy suitable for the proteolysis, purification, and subsequent chemical modification of peptides. We use this resin on an HEK293T cell lysate and perform one-pot proteolysis, capture, and derivatization to survey peptide capture biases from over 40 000 unique peptides from a cellular proteome. We also show that this capture can be reversed in a traceless manner, such that it is amenable for single-molecule proteomics techniques. With this technique, we perform a fluorescent labeling and C-terminal derivatization on a peptide and subject it to fluorosequencing, demonstrating that washing the resin is sufficient to remove excess dyes and other reagents prior to single-molecule protein sequencing.

Unsupervised Idealization of Nano-Electronic Sensors Recordings with Concept Drifts: An Information Theory Approach for Non-Stationary Single-Molecule Data Analysis

Unsupervised Idealization of Nano-Electronic Sensors Recordings with Concept Drifts: An Information Theory Approach for Non-Stationary Single-Molecule Data Analysis

Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.

Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.

Application of Solid-State Nanopore in Protein Detection.

A protein is a kind of major biomacromolecule of life. Its sequence, structure, and content in organisms contains quite important information for normal or pathological physiological process. However, research of proteomics is facing certain obstacles. Only a few technologies are available for protein analysis, and their application is limited by chemical modification or the need for a large amount of sample. Solid-state nanopore overcomes some shortcomings of the existing technology, and has the ability to detect proteins at a single-molecule level, with its high sensitivity and robustness of device. Many works on detection of protein molecules and discriminating structure have been carried out in recent years. Single-molecule protein sequencing techniques based on solid-state nanopore are also been proposed and developed. Here, we categorize and describe these efforts and progress, as well as discuss their advantages and drawbacks.

Single-molecule observation of ATP-independent SSB displacement by RecO in Deinococcus radiodurans.

Deinococcus radiodurans (DR) survives in the presence of hundreds of double-stranded DNA (dsDNA) breaks by efficiently repairing such breaks. RecO, a protein that is essential for the extreme radioresistance of DR, is one of the major recombination mediator proteins in the RecA-loading process in the RecFOR pathway. However, how RecO participates in the RecA-loading process is still unclear. In this work, we investigated the function of drRecO using single-molecule techniques. We found that drRecO competes with the ssDNA-binding protein (drSSB) for binding to the freely exposed ssDNA, and efficiently displaces drSSB from ssDNA without consuming ATP. drRecO replaces drSSB and dissociates it completely from ssDNA even though drSSB binds to ssDNA approximately 300 times more strongly than drRecO does. We suggest that drRecO facilitates the loading of RecA onto drSSB-coated ssDNA by utilizing a small drSSB-free space on ssDNA that is generated by the fast diffusion of drSSB on ssDNA.

Advances in Optical Single-Molecule Detection: En Route to Supersensitive Bioaffinity Assays.

The ability to detect low concentrations of analytes and in particular low‐abundance biomarkers is of fundamental importance, e.g., for early‐stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single‐molecule bioaffinity assays. While many review articles have highlighted the potentials of single‐molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real‐world applications as one should expect. This Review provides a theoretical background on single‐molecule—or better digital—assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single‐molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials.

Wenjing Hong.

"The most significant scientific advance of the last 100 years has been quantum mechanics … What I look for first in a publication is something unexpected and counterintuitive …" Find out more about Wenjing Hong in his Author Profile.

Studying heat shock proteins through single-molecule mechanical manipulation.

Imbalances of cellular proteostasis are linked to ageing and human diseases, including neurodegenerative and neuromuscular diseases. Heat shock proteins (HSPs) and small heat shock proteins (sHSPs) together form a crucial core of the molecular chaperone family that plays a vital role in maintaining cellular proteostasis by shielding client proteins against aggregation and misfolding. sHSPs are thought to act as the first line of defence against protein unfolding/misfolding and have been suggested to act as "sponges" that rapidly sequester these aberrant species for further processing, refolding, or degradation, with the assistance of the HSP70 chaperone system. Understanding how these chaperones work at the molecular level will offer unprecedented insights for their manipulation as therapeutic avenues for the treatment of ageing and human disease. The evolution in single-molecule force spectroscopy techniques, such as optical tweezers (OT) and atomic force microscopy (AFM), over the last few decades have made it possible to explore at thesingle-molecule level the structural dynamics of HSPs and sHSPs and toexamine the key molecular mechanisms underlying their chaperone activities. In this paper, we describe the working principles of OT and AFM and the experimental strategies used to employ these techniques to study molecular chaperones. We then describe the results of some of the most relevant single-molecule manipulation studies on HSPs and sHSPs and discuss how these findings suggest a more complex physiological role for these chaperones than previously assumed.