Single-molecule Methodologies Research Articles

Identification and quantification of within-burst dynamics in singly labeled single-molecule fluorescence lifetime experiments

Single-molecule spectroscopy has revolutionized molecular biophysics and provided means to probe how structural moieties within biomolecules spatially reorganize at different timescales. There are several single-molecule methodologies that probe local structural dynamics in the vicinity of a single dye-labeled residue, which rely on fluorescence lifetimes as readout. Nevertheless, an analytical framework to quantify dynamics in such single-molecule single dye fluorescence bursts, at timescales of microseconds to milliseconds, has not yet been demonstrated. Here, we suggest an analytical framework for identifying and quantifying within-burst lifetime-based dynamics, such as conformational dynamics recorded in single-molecule photo-isomerization-related fluorescence enhancement. After testing the capabilities of the analysis on simulations, we proceed to exhibit within-burst millisecond local structural dynamics in the unbound α-synuclein monomer. The analytical framework provided in this work paves the way for extracting a full picture of the energy landscape for the coordinate probed by fluorescence lifetime-based single-molecule measurements.

Of numbers and movement - understanding transcription factor pathogenesis by advanced microscopy.

ABSTRACTTranscription factors (TFs) are life-sustaining and, therefore, the subject of intensive research. By regulating gene expression, TFs control a plethora of developmental and physiological processes, and their abnormal function commonly leads to various developmental defects and diseases in humans. Normal TF function often depends on gene dosage, which can be altered by copy-number variation or loss-of-function mutations. This explains why TF haploinsufficiency (HI) can lead to disease. Since aberrant TF numbers frequently result in pathogenic abnormalities of gene expression, quantitative analyses of TFs are a priority in the field. In vitro single-molecule methodologies have significantly aided the identification of links between TF gene dosage and transcriptional outcomes. Additionally, advances in quantitative microscopy have contributed mechanistic insights into normal and aberrant TF function. However, to understand TF biology, TF-chromatin interactions must be characterised in vivo, in a tissue-specific manner and in the context of both normal and altered TF numbers. Here, we summarise the advanced microscopy methodologies most frequently used to link TF abundance to function and dissect the molecular mechanisms underlying TF HIs. Increased application of advanced single-molecule and super-resolution microscopy modalities will improve our understanding of how TF HIs drive disease.

Single-molecule analysis of subtelomeres and telomeres in Alternative Lengthening of Telomeres (ALT) cells

BackgroundTelomeric DNA is typically comprised of G-rich tandem repeat motifs and maintained by telomerase (Greider CW, Blackburn EH; Cell 51:887–898; 1987). In eukaryotes lacking telomerase, a variety of DNA repair and DNA recombination based pathways for telomere maintenance have evolved in organisms normally dependent upon telomerase for telomere elongation (Webb CJ, Wu Y, Zakian VA; Cold Spring Harb Perspect Biol 5:a012666; 2013); collectively called Alternative Lengthening of Telomeres (ALT) pathways. By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2.ResultsOur results revealed loci-specific ALT telomere features. For example, while each subtelomere included examples of single molecules with terminal (TTAGGG) n tracts as well as examples of recombinant telomeric single molecules, the ratio of these molecules was subtelomere-specific, ranging from 33:1 (19p) to 1:25 (19q) in U2OS. The Saos-2 cell line shows a similar percentage of recombinant telomeres. The frequency of recombinant subtelomeres of SK-MEL-2 (11%) is about half that of U2OS and Saos-2 (24 and 19% respectively). Terminal (TTAGGG) n tract lengths and heterogeneity levels, the frequencies of telomere signal-free ends, and the frequency and size of retained internal telomere-like sequences (ITSs) at recombinant telomere fusion junctions all varied according to the specific subtelomere involved in a particular cell line. Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were found in all three cell lines; these are in principle capable of templating synthesis of new long telomere tracts via break-induced repair (BIR) long-tract DNA synthesis mechanisms and contributing to the very long telomere tract length and heterogeneity characteristic of ALT cells. Many of longest telomere tracts (both end-telomeres and linear ECTRs) displayed punctate CRISPR/Cas9-dependent (TTAGGG) n labeling patterns indicative of interspersion of stretches of non-canonical telomere repeats.ConclusionIdentifying individual subtelomeres and characterizing linked telomere (TTAGGG) n tract lengths and structural changes using our new single-molecule methodologies reveals the structural consequences of telomere damage, repair and recombination mechanisms in human ALT cells in unprecedented molecular detail and significant differences in different ALT-positive cell lines.

Imaging and Force Spectroscopy of Single Transmembrane Proteins with the Atomic Force Microscope.

The atomic force microscope (AFM) has opened avenues and provided opportunities to investigate biological soft matter and processes ranging from nanometer (nm) to millimeter (mm). The high temporal (millisecond) and spatial (nanometer) resolutions of the AFM are suited for studying many biological processes in their native conditions. The AFM cantilever-aptly termed as a "lab on a tip"-can be used as an imaging tool as well as a handle to manipulate single bonds and proteins. Recent examples have convincingly established AFM as a tool to study the mechanical properties and monitor processes of single proteins and cells with high sensitivity, thus affording insight into important mechanistic details. This chapter specifically focuses on practical and analytical protocols of single-molecule AFM methodologies related to high-resolution imaging and single-molecule force spectroscopy of transmembrane proteins in a lipid bilayer (reconstituted or native). Both these techniques are operator oriented, and require specialized working knowledge of the instrument, theory and practical skills.

Dynamic chromatin technologies: from individual molecules to epigenomic regulation in cells.

The establishment and maintenance of chromatin states involves multiscale dynamic processes integrating transcription factor and multiprotein effector dynamics, cycles of chemical chromatin modifications, and chromatin structural organization. Recent developments in genomic technologies are emerging that are enabling a view beyond ensemble- and time-averaged properties and are revealing the importance of dynamic chromatin states for cell fate decisions, differentiation and reprogramming at the single-cell level. Concurrently, biochemical and single-molecule methodologies are providing key insights into the underlying molecular mechanisms. Combining results from defined in vitro and single-molecule studies with single-cell genomic approaches thus holds great promise for understanding chromatin-based transcriptional memory and cell fate. In this Review, we discuss recent developments in biochemical, single-molecule biophysical and single-cell genomic technologies and review how the findings from these approaches can be integrated to paint a comprehensive picture of dynamic chromatin states.

The fluorescence properties and binding mechanism of SYTOX green, a bright, low photo-damage DNA intercalating agent.

DNA intercalators are widely used in cancer therapeutics, to probe protein-DNA interactions and to investigate the statistical-mechanical properties of DNA. Here, we employ single-molecule fluorescence microscopy, magnetic tweezers, and ensemble-binding assays to investigate the fluorescence properties and binding mechanism of SYTOX green, a DNA labeling dye previously used for staining dead cells and becoming of common use for single-molecule methodologies. Specifically, we show that SYTOX green presents several advantages with respect to other dyes: (1) binds DNA rapidly and with high affinity; (2) has a good signal-to-noise ratio even at low concentrations; (3) exhibits a low photobleaching rate; and (4) induces lower light-induced DNA degradation. Finally, we show that SYTOX green is a DNA intercalator that binds DNA cooperatively with a binding site of 3.5 bp, increasing the DNA length upon binding by 43%, while not affecting its mechanical properties.

Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles.

This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less supercoiling results in more movement, and more supercoiling results in less movement. In contrast to other single-molecule methodologies, the current methodology allows for studying DNA in its naturally supercoiled state with constant linking number and constant writhe. The protocol has potential for use in studying the influence of supercoils on the dynamics of DNA and its associated proteins, e.g., topoisomerase. The procedure takes ~4 weeks.

Unraveling the Mysteries of Chaperone Interactions of the Myosin Head

Unraveling the Mysteries of Chaperone Interactions of the Myosin Head

Visualizing protein movement on DNA at the single-molecule level using DNA curtains

A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively.

A Slippery Walk to the Microtubule-End

A Slippery Walk to the Microtubule-End

Single Molecule Characterization of Alzheimer's Aβ Peptide Interactions with Model and Cell-Derived Membranes

The amyloid plaques found in the brains of Alzheimer's disease patients are a hallmark of this disease and are mostly comprised of the short, 40-42 amino acid, protein fragment known as Aβ, which is derived by the proteolytic processing of the much larger amyloid precursor protein. A major current hypothesis regarding the mechanism of neurotoxicity of Aβ posits that the binding of Aβ to cell membranes induces oligomerization of the peptide leading to the formation of transmembrane pores that compromise the ability of the cell to control intracellular ion concentrations. Work in our laboratory has characterized the mechanism of interaction of Aβ with model phospholipid membranes using calorimetric, electrophysiological and optical spectroscopic techniques. In particular, single molecule methodologies are utilized to monitor the interactions of fluorescently labeled Aβ on lipid membranes. Our results reveal that Aβ interaction with model membranes is characterized by significant heterogeneity in the resulting oligomers, in the corresponding binding enthalpies as well as in the membrane permeabilization potentials. These studies provide detailed insight into the physical chemical basis of Aβ-lipid membrane interaction and its outcome. We are currently expanding our studies to more physiologically relevant systems including membrane blebs and SH-SY5Y neuroblastoma cells. Our data on RBL (rat basophilic leukemia) blebs indicates that both labeled and unlabeled Aβ is capable of forming pores that facilitate the flow of Ca ions as monitored by internalized Ca indicators. We will address the role of specific membranes in Aβ-lipid interactions.

Mechanistic Studies at the Single Molecule Level Reveal the Dynamics of HCV Polymerase Protein in Complex with RNA

Viral RNA-dependent RNA polymerases (RdRp) that belong to the Flaviviridae family, including hepatitis C virus (HCV), are capable of initiating de novo RNA synthesis. The non-structural protein 5B (NS5B) in HCV shows RdRp activity that is required for viral replication. Due to its critical role in life cycle, understanding the mechanism of mRNA synthesis in HCV is fundamental for current drug discovery efforts.We will present our results on studies on the enzymatic activity of the Hepatitis C Virus (HCV) RNA polymerase protein. Our studies, conducted with state-of-the-art fluorescence single molecule methodologies, aim to elucidate the dynamics of key protein/nucleic acid complexes.Our work was conducted on DNA:RNA templates labeled with Cy5/Cy3 (Acceptor/Donor) fluorophores capable of undergoing Forster Resonance Energy Transfer (FRET). Binding of NS5B caused a significant increase in FRET. The SM-FRET studies on RNA-protein complexes revealed protein dynamics occurring with time scales of a few seconds. These dynamics change with the RNA template length, and with the presence of complementary DNA strands.Taken together, our single molecule studies provide for the first time direct evidence on the polymerase-substrate binding process and the effect of template length on protein dynamics.

Single-Molecule FRET

Single molecule studies have revolutionized our understanding of how biomolecules work. The ability to watch one molecule at a time reveals not only the average properties detected in ensemble measurements, but can yield the entire distribution of relevant properties, including subpopulations and rare events. Single molecule studies also reveal the time evolution of biochemical reactions,which, if asynchronous, are not observable via ensemble measurements. In the 1970s, for example, the development of the patch-clamp technique enabled the electrical current through a single ion channel to be measured, revealing many properties, including that ion channels are on/off devices. More recently, single molecule techniques applicable beyond ion channels have been developed. Methods such as optical (or magnetic) tweezers and scanning force microscopy are making it possible to follow, in real-time, the movements, forces and strains that develop during the course of a reaction (reviewed in [1]). These methods can be used to measure directly the forces that hold together molecular structures. New developments in single molecule fluorescence methodology are also broadening the scope of single molecule studies. Single biomolecules are labeled with an extrinsic fluorophore, and the fluorescent properties of the fluorophore can report on conformational changes occurring in the biomolecules. For example, intensity fluctuations of a single or small number of molecules can also measure reaction rates as well as the number of molecules present. Single enzyme reaction kinetics, for example, were measured in this way and found to have both static and dynamic disorder, as well as a rate which depended on the enzyme's history, properties which were undetectable via ensemble methods [2]. In addition, single molecule fluorescence polarization can be used to infer local rotation or changes in flexibility of biomolecules (reviewed in [3, 4]). Perhaps the most general approach is the use of two fluorophores rather than one, in the form of fluorescence resonance energy transfer (FRET). In FRET, a “donor” dye transfers energy to an “acceptor” dye, and the donor intensity decreases and the acceptor intensity increases as the two dyes approach each other (reviewed in [5]). Importantly, it is sensitive to sub-nanometer distance changes in the range of 20-80 A, a sensitivity and range well suited for studying biomolecules. Excitation energy of the donor is transferred to the acceptor via an induced-dipole, induced-dipole interaction. The efficiency of energy transfer, E, is given by: where R is the distance between the donor and acceptor and R0 is the distance at which 50% of the energy is transferred and is a function of the properties of the dyes. Because of this strong distance dependence, structural changes of biomolecules or relative motion and interaction between two different molecules can be detected via FRET. These conformational changes are often difficult to synchronize or too rare to detect using ensemble FRET studies. Single-molecule FRET (smFRET) overcomes these difficulties, creating new opportunities to probe structural changes of biological molecules during biological events in real time. SmFRET is also relatively insensitive to incomplete labeling of host molecule with donor and acceptor because donor-only species simply show up as an FRET = 0 peak while acceptor-only species are excited only very weakly by the excitation light if the probes are selected with a large spectral separation. Unlike other single-molecule methodologies measuring forces (optical or magnetic tweezers and atomic force microscopy) [1], single-molecule FRET is sensitive to the internal motion or arrangement of host biological molecules in its center of mass frame; therefore it does not suffer from Brownian motion of the whole molecule. In addition, it can be applied to a much broader class of biological systems, because it does not require force-generating molecules. It is especially powerful in capturing rare, transient events and in dissecting complex multi-step processes. Since our first demonstration of single molecule FRET, there have been a number of biological applications (and see review in [4, 6]). Ever expanding list of biological systems that have been studied by single molecule FRET includes DNA rulers [7-9], Staphylococcal nuclease [10], biotin-Streptavidin [11], GCN4 peptides [12], a-tropomyosin [13], S15 binding RNA junction [14], Tetrahymena ribozyme [15], calmodulin [16], and Rep helicase (Ha et al, unpublished data). There are already excellent reviews on the existing single molecule FRET works [4, 6] and a review focused on practical issues regarding smFRET implementation is available [17].

Single-Molecule FRET

Single molecule studies have revolutionized our understanding of how biomolecules work. The ability to watch one molecule at a time reveals not only the average properties detected in ensemble measurements, but can yield the entire distribution of relevant properties, including subpopulations and rare events. Single molecule studies also reveal the time evolution of biochemical reactions,which, if asynchronous, are not observable via ensemble measurements. In the 1970s, for example, the development of the patch-clamp technique enabled the electrical current through a single ion channel to be measured, revealing many properties, including that ion channels are on/off devices. More recently, single molecule techniques applicable beyond ion channels have been developed. Methods such as optical (or magnetic) tweezers and scanning force microscopy are making it possible to follow, in real-time, the movements, forces and strains that develop during the course of a reaction (reviewed in [1]). These methods can be used to measure directly the forces that hold together molecular structures. New developments in single molecule fluorescence methodology are also broadening the scope of single molecule studies. Single biomolecules are labeled with an extrinsic fluorophore, and the fluorescent properties of the fluorophore can report on conformational changes occurring in the biomolecules. For example, intensity fluctuations of a single or small number of molecules can also measure reaction rates as well as the number of molecules present. Single enzyme reaction kinetics, for example, were measured in this way and found to have both static and dynamic disorder, as well as a rate which depended on the enzyme's history, properties which were undetectable via ensemble methods [2]. In addition, single molecule fluorescence polarization can be used to infer local rotation or changes in flexibility of biomolecules (reviewed in [3, 4]). Perhaps the most general approach is the use of two fluorophores rather than one, in the form of fluorescence resonance energy transfer (FRET). In FRET, a “donor” dye transfers energy to an “acceptor” dye, and the donor intensity decreases and the acceptor intensity increases as the two dyes approach each other (reviewed in [5]). Importantly, it is sensitive to sub-nanometer distance changes in the range of 20-80 Å, a sensitivity and range well suited for studying biomolecules. Excitation energy of the donor is transferred to the acceptor via an induced-dipole, induced-dipole interaction. The efficiency of energy transfer, E, is given by: where R is the distance between the donor and acceptor and R0 is the distance at which 50% of the energy is transferred and is a function of the properties of the dyes. Because of this strong distance dependence, structural changes of biomolecules or relative motion and interaction between two different molecules can be detected via FRET. These conformational changes are often difficult to synchronize or too rare to detect using ensemble FRET studies. Single-molecule FRET (smFRET) overcomes these difficulties, creating new opportunities to probe structural changes of biological molecules during biological events in real time. SmFRET is also relatively insensitive to incomplete labeling of host molecule with donor and acceptor because donor-only species simply show up as an FRET = 0 peak while acceptor-only species are excited only very weakly by the excitation light if the probes are selected with a large spectral separation. Unlike other single-molecule methodologies measuring forces (optical or magnetic tweezers and atomic force microscopy) [1], single-molecule FRET is sensitive to the internal motion or arrangement of host biological molecules in its center of mass frame; therefore it does not suffer from Brownian motion of the whole molecule. In addition, it can be applied to a much broader class of biological systems, because it does not require force-generating molecules. It is especially powerful in capturing rare, transient events and in dissecting complex multi-step processes. Since our first demonstration of single molecule FRET, there have been a number of biological applications (and see review in [4, 6]). Ever expanding list of biological systems that have been studied by single molecule FRET includes DNA rulers [7-9], Staphylococcal nuclease [10], biotin-Streptavidin [11], GCN4 peptides [12], a-tropomyosin [13], S15 binding RNA junction [14], Tetrahymena ribozyme [15], calmodulin [16], and Rep helicase (Ha et al, unpublished data). There are already excellent reviews on the existing single molecule FRET works [4, 6] and a review focused on practical issues regarding smFRET implementation is available [17].

Probing Single Molecules in Single Living Cells

Direct observation of single molecules and single molecular events inside living cells could dramatically improve our understanding of basic cellular processes (e.g., signal transduction and gene transcription) as well as improving our knowledge on the intracellular transport and fate of therapeutic agents (e.g., antisense RNA and gene therapy vectors). However, a key remaining question is whether single-molecule methodologies could be developed to study complex molecular processes in living cells. in contrast to clean and well-controlled conditions in-vitro, the intracellular environment contains a broad collection of biological macromolecules and fluorescent materials such as porphyrins and flavins. This complex environment is known to produce intense background fluorescence, commonly known as autofluorescence. Thus, a major concern is that this intracellular background could overwhelm the relatively weak signals arising from single molecules.We demonstrate that fluorescence detection of single molecules can be achieved by tightly focusing a laser beam into a living cell (see Figure 1). The observed background fluorescence is indeed higher than that in-vitro (e.g., pure biological buffer), but this background is continuous and stable, and does not significantly interfere with the measurement of single-molecule photon bursts. Specifically, we report single-molecule results on three types of extrinsic fluorescent molecules in cultured human HeLa cells (a cervical cancer cell line).