Single-molecule Methods Research Articles

Quantitative single-molecule analysis of assembly and Ca2+-dependent disassembly of synaptotagmin oligomers on lipid bilayers

Synaptotagmin-1 (Syt-1) self-assembles into ring-like oligomers, and genetic and biochemical evidence suggest that oligomerization is needed to clamp synaptic vesicles and stabilize them for Ca2+-evoked release. However, oligomerization has not yet been demonstrated on lipid bilayers or studied in quantitative biophysical terms. Here we utilize single-molecule imaging methods to monitor the assembly and disassembly of oligomeric clusters of Syt-1 on lipid bilayers in real-time. Syt-1 assembled into two distinct classes of oligomers, small (5 ± 2 subunits) and large (15 ± 2 subunits). Each class assembled at a constant kon that was always proportional to its ultimate size, but both classes disassembled at the same unit rate (koff) independent of its size. Both large and small oligomers explosively disassembled when Ca2+ was added. The F349A mutation in the Syt-1 nearly eliminates the large class of oligomers but does not reduce the small class. Altogether, the physical-chemical properties of Syt-1 oligomers meet or exceed the physiologic requirements to function as such a clamp.

Quantitative analysis of transferrin uptake into living cells at single-molecule level

Endocytosis, apart from providing a mechanism for cells to take up nutrients, is the principal route of entry for many nanomedicines. The evaluation of therapeutic delivery efficiency without providing quantitative parameters, like the number of molecules entering the cell and the time of their entry, is very limited. Despite advances in single-molecule methods for in-cell quantitative measurements, they have not become widely used in the study of endocytosis. Their application, however, is challenging owing to the appropriate experimental design and applicable analysis methods. Here, by using an integrated approach based on the multidimensional time-correlated single-photon counting (TCSPC) technique, we demonstrate the quantitative method to measure the time- and concentration-dependent uptake of TRITC-transferrin into living cells with single-molecule sensitivity. The methodology opens possibilities for quantitatively assessing the delivery efficiency of fluorescent molecules into living cells.

Nuclease-induced stepwise photodropping (NISP) to precisely investigate single-stranded DNA degradation behaviors of exonucleases and endonucleases.

Here, we employed a fluorescence-based single molecule method called nuclease-induced stepwise photodropping (NISP) to measure in realtime the DNA degradation mediated by mitochondrial genome maintenance exonuclease 1 (MGME1), a bidirectional single-stranded DNA (ssDNA)-specific exonuclease. The method detects a stepwise decrease in fluorescence signals from Cy3 fluorophores labeled on an immobilized DNA substrate. Using NISP, we successfully determined the DNA degradation rates of 6.3±0.4and 2.0±0.1 nucleotides (nt) s-1 for MGME1 in the 5'-to-3' and 3'-to-5' directions, respectively. These results provide direct evidence of the stronger 5' directionality of MGME1, consistent with its established role in mitochondrial DNA maintenance. Importantly, when we employed NISP to investigate mung bean nuclease, an ss-specific endonuclease, we observed a markedly different NISP pattern, suggesting a distributive cleavage activity of the enzyme. Furthermore, we applied NISP to determine the ssDNA degradation behavior of the double-stranded-specific exonuclease, λexonuclease. These findings underscore the capability of NISP to accurately and reliably measure the degradation of ssDNA by both exo- and endonucleases. Here, we demonstrate NISP as a powerful tool for investigating the ssDNA degradation behavior of nucleases at the single-molecule level.

Protection of the Telomeric Junction by the Shelterin Complex.

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats, while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared with POT1 alone by bridging single- and double-stranded telomeric tracts.

Rate-Engineered Plasmon-Enhanced Fluorescence for Real-Time Microsecond Dynamics of Single Biomolecules.

Single-molecule fluorescence has revealed a wealth of biochemical processes but does not give access to submillisecond dynamics involved in transient interactions and molecular dynamics. Here we overcome this bottleneck and demonstrate record-high photon count rates of >107 photons/s from single plasmon-enhanced fluorophores. This is achieved by combining two conceptual novelties: first, we balance the excitation and decay rate enhancements by the antenna's volume, resulting in maximum fluorescence intensity. Second, we enhance the triplet decay rate using a multicomponent surface chemistry that minimizes microsecond blinking. We demonstrate applications to two exemplary molecular processes: we first reveal transient encounters and hybridization of DNA with a 1 μs temporal resolution. Second, we exploit the field gradient around the nanoparticle as a molecular ruler to reveal microsecond intramolecular dynamics of multivalent complexes. Our results pave the way toward real-time microsecond studies of biochemical processes using an implementation compatible with existing single-molecule fluorescence methods.

Two near-chromosomal-level genomes of globally-distributed Macroascomycete based on single-molecule fluorescence and Hi-C methods

Discinaceae holds significant importance within the Pezizales, representing a prominent group of macroascomycetes distributed globally. However, there is a dearth of genomic studies focusing on this family, resulting in gaps in our understanding of its evolution, development, and ecology. Here we utilized state-of-the-art genome assembly methodologies, incorporating third-generation single-molecule fluorescence and Hi-C-assisted methods, to elucidate the genomic landscapes of Gyromitra esculenta and Paragyromitra xinjiangensis. The genome sizes of two species were determined to be 47.10 Mb and 48.20 Mb, with 23 and 22 scaffolds, respectively. 10,438 and 11,469 coding proteins were identified, with functional annotations encompassing over 96.47% and 94.40%, respectively. Assessment of completeness using BUSCO revealed that 98.71% and 98.89% of the conserved proteins were identified. The application of comparative genomic technology has helped in identifying traits associated with of heterothallic life cycle traits and elucidating unique patterns of chromosomal evolution. Additionally, we identified potential saprotrophic nutritional modes and systematic phylogenetic relationships between the two species. Therefore, this study provides crucial genomic insights into the evolution, nutritional type, and ecological roles of species within the Pezizales.

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers.

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Tracing Homopolymers in Oikopleura dioica's Mitogenome.

Oikopleura dioica is a planktonic tunicate (Appendicularia class) found extensively across the marine waters of the globe. The genome of a single male individual collected from Okinawa, Japan was sequenced using the single-molecule PacBio Hi-Fi method and assembled with NOVOLoci. The mitogenome is 39,268 bp long, featuring a large control region of around 22,000 bp. We annotated the proteins atp6, cob, cox1, cox2, cox3, nad1, nad4, and nad5, and found one more open reading frame that did not match any known gene. This study marks the first complete mitogenome assembly for an appendicularian, and reveals that A and T homopolymers cumulatively account for nearly half of its length. This reference sequence will be an asset for environmental DNA and phylogenetic studies.

Ultrafast Super-Resolution Imaging Exploiting Spontaneous Blinking of Static Excimer Aggregates.

Single-molecule localization methods have been popularly exploited to obtain super-resolved images of biological structures. However, the low blinking frequency of randomly switching emission states of individual fluorophores greatly limits the imaging speed of single-molecule localization microscopy (SMLM). Here we present an ultrafast SMLM technique exploiting spontaneous fluorescence blinking of cyanine dye aggregates confined to DNA framework nanostructures. The DNA template guides the formation of static excimer aggregates as a "light-harvesting nanoantenna", whereas intermolecular excitation energy transfer (EET) between static excimers causes collective ultrafast fluorescence blinking of fluorophore aggregates. This DNA framework-based strategy enables the imaging of DNA nanostructures with 12.5-fold improvement in speed compared to conventional SMLM. Further, we demonstrate the use of this strategy to track the movement of super-resolved DNA nanostructures for over 20 min in a microfluidic system. Thus, this ultrafast SMLM holds great potential for revealing the dynamic processes of biomacromolecules in living cells.

Different methods for single molecule detection

Abstract The molecule is the smallest unit that can exist independently and maintain the physical and chemical properties of the substance, so single molecule detection is a critical technology in biology, chemistry, and other disciplines. Today, different instruments and methods for single-molecule detection exist. Each single molecule detection method’s principle, operation, and application scope differ. Systematic analysis and summary are needed. This research will outline the principles and recent achievements of several single-molecule detection methods, from microscopes to spectrometers and biosensors. The development of atomic force microscopy (AFM) is mainly introduced in microscopy. First, the fundamentals of AFM will be introduced, followed by high-speed atomic AFM (Hs-AFM) and its integrated green laser irradiation of single molecules for dynamics, Later, we will introduce the recently developed Localization AFM (LAFM) with higher resolution and the Single-molecule localization microscopy (SMLM) with similar principle. Raman spectroscopy and Raman imaging are mainly introduced in spectroscopy. In the last aspect of biosensors, the principle and development of nanopore and enzyme-linked immunosorbent assay (ELISA) are mainly introduced.

Cholesterol inhibits assembly and activation of the EphA2 receptor.

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

Quantifying Microsecond Solution-Phase Conformational Dynamics of a DNA Hairpin at the Single-Molecule Level.

Quantifying the rapid conformational dynamics of biological systems is fundamental to understanding the mechanism. However, biomolecules are complex, often containing static and dynamic heterogeneity, thus motivating the use of single-molecule methods, particularly those that can operate in solution. In this study, we measure microsecond conformational dynamics of solution-phase DNA hairpins at the single-molecule level using an anti-Brownian electrokinetic (ABEL) trap. Different conformational states were distinguished by their fluorescence lifetimes, and kinetic parameters describing transitions between these states were determined using two-dimensional fluorescence lifetime correlation (2DFLCS) analysis. Rather than combining fluorescence signals from the entire data set ensemble, long observation times of individual molecules allowed ABEL-2DFLCS to be performed on each molecule independently, yielding the underlying distribution of the system's kinetic parameters. ABEL-2DFLCS on the DNA hairpins resolved an underlying heterogeneity of fluorescence lifetimes and provided signatures of two-state exponential dynamics with rapid (<millisecond) transition times between states without observation of the substantially stretched exponential kinetics that had been observed in previous measurements on diffusing molecules. Numerical simulations were performed to validate the accuracy of this technique and the effects the underlying heterogeneity has on the analysis. Finally, ABEL-2DFLCS was performed on a mixture of hairpins and used to resolve their kinetic data.

Using Förster Resonance Energy Transfer (FRET) to Understand the Ubiquitination Landscape.

Förster resonance energy transfer (FRET) is a fluorescence technique that allows quantitative measurement of protein interactions, kinetics and dynamics. This review covers the use of FRET to study the structures and mechanisms of ubiquitination and related proteins. We survey FRET assays that have been developed where donor and acceptor fluorophores are placed on E1, E2 or E3 enzymes and ubiquitin (Ub) to monitor steady-state and real-time transfer of Ub through the ubiquitination cascade. Specialized FRET probes placed on Ub and Ub-like proteins have been developed to monitor Ub removal by deubiquitinating enzymes (DUBs) that result in a loss of a FRET signal upon cleavage of the FRET probes. FRET has also been used to understand conformational changes in large complexes such as multimeric E3 ligases and the proteasome, frequently using sophisticated single molecule methods. Overall, FRET is a powerful tool to help unravel the intricacies of the complex ubiquitination system.

Polymerization mechanism of the Candida albicans virulence factor candidalysin

Candida albicans is a commensal fungus that can cause epithelial infections and life-threatening invasive candidiasis. The fungus secretes candidalysin (CL), a peptide that causes cell damage and immune activation by permeation of epithelial membranes. The mechanism of CL action involves strong peptide assembly into polymers in solution. The free ends of linear CL polymers can join, forming loops that become pores upon binding to membranes. CL polymers constitute a therapeutic target for candidiasis, but little is known about CL self-assembly in solution. Here, we examine the assembly mechanism of CL in the absence of membranes using complementary biophysical tools, including a new fluorescence polymerization assay, mass photometry, and atomic force microscopy. We observed that CL assembly is slow, as tracked with the fluorescent marker C-laurdan. Single-molecule methods showed that CL polymerization involves a convolution of four processes. Self-assembly begins with the formation of a basic subunit, thought to be a CL octamer that is the polymer seed. Polymerization proceeds via addition of octamers, and as polymers grow they can curve and form loops. Alternatively, secondary polymerization can occur and cause branching. Interplay between the different rates determines the distribution of CL particle types, indicating a kinetic control mechanism. This work elucidates key physical attributes underlying CL self-assembly which may eventually evoke pharmaceutical development.

Pulsed-Interleaved-Excitation Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy.

We report on pulsed-interleaved-excitation two-dimensional fluorescence lifetime correlation spectroscopy (PIE 2D FLCS) to study biomolecular structural dynamics with high sensitivity and high time resolution using Förster resonance energy transfer (FRET). PIE 2D FLCS is an extension of 2D FLCS, which is a unique single-molecule fluorescence method that uses fluorescence lifetime information to distinguish different fluorescence species in equilibrium and resolves their interconversion dynamics with a submicrosecond time resolution. Because 2D FLCS has used only a single-color excitation so far, it was difficult to distinguish a very low-FRET (or zero-FRET) species from only donor-labeled species. We overcome this difficulty by implementing the PIE scheme (i.e., alternate excitation of the donor and acceptor dyes using two temporally interleaved excitations with different colors) to 2D FLCS, realizing two-color excitation and two-color fluorescence detection in 2D FLCS. After proof-of-principle PIE 2D FLCS analysis on the photon data synthesized with Monte Carlo simulation, we apply PIE 2D FLCS to a DNA-hairpin sample and show that this method readily distinguishes four fluorescent species, i.e., high-FRET, low-FRET, and two single-dye-labeled species. In addition, we show that PIE 2D FLCS can also quantitatively evaluate the contributions of the donor-acceptor spectral crosstalk, which often appears as artifacts in FRET studies and degrades the information obtained.

Features of the DNA Escherichia coli RecN interaction revealed by fluorescence microscopy and single-molecule methods

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.

Subunit Communication within Dimeric SF1 DNA Helicases

Monomers of the Superfamily (SF) 1 helicases, E. coli Rep and UvrD, can translocate directionally along single stranded (ss) DNA, but must be activated to function as helicases. In the absence of accessory factors, helicase activity requires Rep and UvrD homo-dimerization. The ssDNA binding sites of SF1 helicases contain a conserved aromatic amino acid (Trp250 in Rep and Trp256 in UvrD) that stacks with the DNA bases. Here we show that mutation of this Trp to Ala eliminates helicase activity in both Rep and UvrD. Rep(W250A) and UvrD(W256A) can still dimerize, bind DNA, and monomers still retain ATP-dependent ssDNA translocase activity, although with ∼10-fold lower rates and lower processivities than wild type monomers. Although neither wtRep monomers nor Rep(W250A) monomers possess helicase activity by themselves, using both ensemble and single molecule methods, we show that helicase activity is achieved upon formation of a Rep(W250A)/wtRep hetero-dimer. An ATPase deficient Rep monomer is unable to activate a wtRep monomer indicating that ATPase activity is needed in both subunits of the Rep hetero-dimer. We find the same results with E. coli UvrD and its equivalent mutant (UvrD(W256A)). Importantly, Rep(W250A) is unable to activate a wtUvrD monomer and UvrD(W256A) is unable to activate a wtRep monomer indicating that specific dimer interactions are required for helicase activity. We also demonstrate subunit communication within the dimer by virtue of Trp fluorescence signals that only are present within the Rep dimer, but not the monomers. These results bear on proposed subunit switching mechanisms for dimeric helicase activity.

Investigation of metal ion binding biomolecules one molecule at a time.

Metal ions can perform multiple roles ranging from regulatory to structural and are crucial for cell function. While some metal ions like Na+ are ubiquitously present at high concentrations, other ions, especially Ca2+ and transition metals, such as Zn2+ or Cu+/2+ are regulated. The concentrations above or below the physiological range cause severe changes in the behavior of biomolecules that bind them and subsequently affect the cell wellbeing. This has led to the development of specialized protocols to study metal ion binding biomolecules in bulk conditions that mimic the cell environment. Recently, there is growing evidence of influence of post-transcriptional and post-translational modifications on the affinity of the metal ion binding sites. However, such targets are difficult to obtain in amounts required for classical biophysical experiments. Single molecule techniques have revolutionized the field of biophysics, molecular and structural biology. Their biggest advantage is the ability to observe each molecule's interaction independently, without the need for synchronization. An additional benefit is its extremely low sample consumption. This feature allows characterization of designer biomolecules or targets obtained coming from natural sources. All types of biomolecules, including proteins, DNA and RNA were characterized using single molecule methods. However, one group is underrepresented in those studies. These are the metal ion binding biomolecules. Single molecule experiments often require separate optimization, due to extremely different concentrations used during the experiments. In this review we focus on single molecule methods, such as single molecule FRET, nanopores and optical tweezers that are used to study metal ion binding biomolecules. We summarize various examples of recently characterized targets and reported experimental conditions. Finally, we discuss the potential promises and pitfalls of single molecule characterization on metal ion binding biomolecules.

Full-Length Single Protein Molecules Tracking and Counting in Thin Silicon Channels.

Emerging single-molecule protein sensing techniques are ushering in a transformative era in biomedical research. Nevertheless, challenges persist in realizing ultra-fast full-length protein sensing, including loss of molecular integrity due to protein fragmentation, biases introduced by antibodies affinity, identification of proteoforms, and low throughputs. Here, a single-molecule method for parallel protein separation and tracking is introduced, yielding multi-dimensional molecular properties used for their identification. Proteins are tagged by chemo-selective dual amino-acid specific labels and are electrophoretically separated by their mass/charge in custom-designed thin silicon channel with subwavelength height. This approach allows analysis of thousands of individual proteins within a few minutes by tracking their motion during the migration. The power of the method is demonstrated by quantifying a cytokine panel for host-response discrimination between viral and bacterial infections. Moreover, it is shown that two clinically-relevant splice isoforms of Vascular endothelial growth factor (VEGF) can be accurately quantified from human serum samples. Being non-destructive and compatible with full-length intact proteins, this method opens up ways for antibody-free single-protein molecule quantification.

Studies of DNA breathing in exciton-coupled (iCy3)2 dimer-labeled DNA constructs by polarization-sweep single-molecule fluorescence (PS-SMF) microscopy.

Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA 'breathing') play a central role in the assembly of protein-DNA complexes. We present a single-molecule fluorescence method to sensitively measure the local conformational fluctuations of exciton-coupled cyanine [(iCy3)2] dimer-labeled DNA fork constructs in which the dimer probes are placed at varying positions relative to the DNA fork junction. These systems exhibit spectroscopic signals that are sensitive to the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probe label positions. The (iCy3)2 dimer has one symmetric (+) and one anti-symmetric (-) exciton with respective transition dipole moments oriented perpendicular to one another. We excite single molecule samples using a continuous-wave, linearly polarized laser with its polarization direction rotated at a frequency of 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and anti-symmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the signal at the photon-counting level provides information about the distribution of local conformations and conformational dynamics. We analyze our data using a kinetic network model, which we use to parametrize the free energy surface of the system. In addition to observing DNA breathing at and near ss-dsDNA junctions, the approach can be used to study the effects of proteins that bind and function at these sites.