Single-molecule Fluorescence Methods Research Articles

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.

Studies of DNA 'Breathing' by Polarization-Sweep Single-Molecule Fluorescence Microscopy of Exciton-Coupled (iCy3)2 Dimer-Labeled DNA Fork Constructs.

Local fluctuations of the sugar-phosphate backbones and bases of DNA (often called DNA 'breathing') play a variety of critical roles in controlling the functional interactions of the DNA genome with the protein complexes that regulate it. Here, we present a single-molecule fluorescence method that we have used to measure and characterize such conformational fluctuations at and near biologically important positions in model DNA replication fork constructs labeled with exciton-coupled cyanine [(iCy3)2] dimer probes. Previous work has shown that the constructs that we tested here exhibit a broad range of spectral properties at the ensemble level, and these differences can be structurally and dynamically interpreted using our present methodology at the single-molecule level. The (iCy3)2 dimer has one symmetric (+) and one antisymmetric (-) exciton, with the respective transition dipole moments oriented perpendicular to one another. We excite single-molecule samples using a continuous-wave linearly polarized laser, with the polarization direction continuously rotated at the frequency of 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and antisymmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the modulated signal provides information about the distribution of local conformations and the conformational interconversion dynamics of the (iCy3)2 probe. We find that at most construct positions that we examined, the (iCy3)2 dimer-labeled DNA fork constructs can adopt four topologically distinct conformational macrostates. These results suggest that in addition to observing DNA breathing at and near ss-dsDNA junctions, our new methodology should be useful to determine which of these pre-existing macrostates are recognized by, bind to, and are stabilized by various genome-regulatory proteins.

Methods to investigate nucleosome structure and dynamics with single-molecule FRET.

The nucleosome is the fundamental building block of chromatin. Changes taking place at the nucleosome level are the molecular basis of chromatin transactions with various enzymes and factors. These changes are directly and indirectly regulated by chromatin modifications such as DNA methylation and histone post-translational modifications including acetylation, methylation, and ubiquitylation. Nucleosomal changes are often stochastic, unsynchronized, and heterogeneous, making it very difficult to monitor with traditional ensemble averaging methods. Diverse single-molecule fluorescence approaches have been employed to investigate the structure and structural changes of the nucleosome in the context of its interactions with various enzymes such as RNA Polymerase II, histone chaperones, transcription factors, and chromatin remodelers. We utilize diverse single-molecule fluorescence methods to study the nucleosomal changes accompanying these processes, elucidate the kinetics of these processes, and eventually learn the implications of various chromatin modifications in directly regulating these processes. The methods include two- and three-color single-molecule fluorescence resonance energy transfer (FRET), single-molecule fluorescence correlation spectroscopy, and fluorescence (co-)localization. Here we report the details of the two- and three-color single-molecule FRET methods we currently use. This report will help researchers design their single-molecule FRET approaches to investigating chromatin regulation at the nucleosome level.

Dynamics of ribosome scanning during translation initiation.

Faithful recognition of the mRNA start codon at the initial stage of translation is an essential process during regulation of gene expression. For this purpose, eukaryotes utilize a 43S pre-initiation complex (PIC), which moves along the mRNA 5′ untranslated region (UTR) to locate the start codon; this movement is termed “scanning” and is thought to proceed linearly with significantly processive 5′-to-3′ directionality. Although the scanning model was proposed many decades ago, the exact molecular mechanism of the motion, and how it may vary between different mRNAs and under various cellular conditions, remain unclear due to the highly dynamic nature of the process. We developed a multi-color single molecule fluorescence method to observe PIC dynamics in real time on the translation initiation timescale. Using this technology, we aim to directly measure the rate of scanning on full-length yeast mRNA, in order to understand the dynamics of PIC movement along different UTR lengths and structural contexts. Furthermore, we seek to parse the roles of key initiation factors and their biochemical activities in the scanning mechanism. Our data provide direct insights into this vital process in translational control.

Using single molecule polarization-sweep spectroscopy to monitor site-specific fluctuations of internal (iCy3)2 dimer-labeled DNA constructs at ss-ds DNA junctions.

Local fluctuations of the sugar-phosphate backbones of DNA (a form of DNA ‘breathing’) play key roles in protein-DNA assembly and enzymatic function. In this work, we monitored spectroscopic signals from single-molecules of DNA fork constructs labeled with two cyanine optical probes [(iCy3)2], which were rigidly inserted into the sugar-phosphate backbones at opposite positions of complementary single-strands. We thus studied the local conformational fluctuations of the DNA sugar-phosphate backbones at specific (iCy3)2 dimer-labeled positions relative to a single-stranded (ss) double-stranded (ds) DNA junction. These experiments are based on a newly-developed single-molecule spectroscopic method that uses a linearly polarized continuous wave laser in which the polarization direction is continuously rotated at radio frequencies. The emitted fluorescence from the single-molecule sample contains information about conformational changes of the exciton-coupled (iCy3)2 dimer probe, which monitor conformational fluctuations of the vicinal DNA sugar-phosphate backbones. Our results indicate that the local conformation of the DNA sugar-phosphate backbones at positions near ss-dsDNA junctions adopts four topologically-relevant macrostates. The probability that a particular macrostate is occupied depends on backbone position. We apply a kinetic network approach to interpret our observations of DNA backbone fluctuations at and near ss-dsDNA junctions, and we propose conformational assignments based on exciton-coupling models for the macrostates that we observe. This polarization-sweep single-molecule fluorescence method and analysis can be applied to study protein binding and macromolecular function at and near ss-dsDNA junctions, thus providing site-specific information about the free energy landscapes at various functionally relevant positions within such macromolecular complexes.

Supramolecular complexes involved in the coordination of transcription and translation.

Single particle cryo-electron microscopy (cryo-EM) allows us to obtain structural information of increasingly complex and dynamic assemblies. The molecular machines, which carry out gene expression, have been studied in isolation but are often organized in supramolecular complexes to coordinate their functions and regulate each other. Furthermore, new functions emerge as a consequence of this organization, which cannot be easily predicted from structures of the individual machineries. A prominent example is the translation of prokaryotic messenger RNAs by the ribosome, which initiates while they are still being transcribed by RNA polymerase (RNAP). The pioneering ribosome can approach RNAP, forming a supramolecular complex known as the expressome. We previously obtained a series of cryo-EM structures representing decoupled, coupled and collided expressome states, which allowed us to propose roles for coupling of RNAP and the ribosome and provided a framework for studies on the coordination of transcription and translation in bacteria. However, mutual coordination is not restricted to the elongation phase of translation because RNAP can also stimulate translation initiation. We used in vitro single-molecule fluorescence methods and cryo-EM to investigate how translation initiation can be promoted by RNAP. RNAP accelerates binding of the 30S ribosomal subunit to mRNA and this requires ribosomal protein S1 (bS1). The structure of a ribosome-RNAP complex revealed RNAP and bS1 can cooperatively deliver the nascent mRNA for Shine-Dalgarno (SD) duplex formation. Additional structural models showed a reorientation of the SD duplex accompanies the transition from the docking of an mRNA to its accommodation in the decoding center. Thus, transcription-translation coupling can initiate by direct contact between gene expression complexes through bS1.

Single-molecule fluorescence methods for protein biomarker analysis.

Proteins have been considered key building blocks of life. In particular, the protein content of an organism and a cell offers significant information for the in-depth understanding of the disease and biological processes. Single-molecule protein detection/sequencing tools will revolutionize clinical (proteomics) research, offering ultrasensitivity for low-abundance biomarker (protein) detection, which is important for the realization of early-stage disease diagnosis and single-cell proteomics. This improved detection/measurement capability delivers new sets of techniques to explore new frontiers and address important challenges in various interdisciplinary areas including nanostructured materials, molecular medicine, molecular biology, and chemistry. Importantly, fluorescence-based methods have emerged as indispensable tools for single protein detection/sequencing studies, providing a higher signal-to-noise ratio (SNR). Improvements in fluorescent dyes/probes and detector capabilities coupled with advanced (image) analysis strategies have fueled current developments for single protein biomarker detections. For example, in comparison to conventional ELISA (i.e., based on ensembled measurements), single-molecule fluorescence detection is more sensitive, faster, and more accurate with reduced background, high-throughput, and so on. In comparison to MS sequencing, fluorescence-based single-molecule protein sequencing can achieve the sequencing of peptides themselves with higher sensitivity. This review summarizes various typical single-molecule detection technologies including their methodology (modes of operation), detection limits, advantages and drawbacks, and current challenges with recent examples. We describe the fluorescence-based single-molecule protein sequencing/detection based on five kinds of technologies such as fluorosequencing, N-terminal amino acid binder, nanopore light sensing, and DNA nanotechnology. Finally, we present our perspective for developing high-performance fluorescence-based sequencing/detection techniques.

Rotation manipulation of single-molecule magnetic trapping and gene transcription regulation dynamics

Gene transcription regulation is a key step for gene expression in all organisms and responsible for the transmission of genetic information and genome integrity. As one of the most important mechanisms in gene transcription, an RNA polymerase (RNAP) specifically interacts with and unwinds genome DNA to form a transcription bubble where a nascent RNA transcript is polymerized, taking one of the unwound DNA strands as its template. The RNAP translocates along the DNA to transcribe the whole gene by carrying the transcription bubble. In such a way, an RNAP completes its biological task of gene expression by physically acting as a molecular machinery. Thus, an RNAP molecule can be considered as a research object for physicists who are willing to uncover the mechanisms of life processes in a physical view. To achieve this, single-molecule method has been invented and used widely. As one of these methods, single-molecule magnetic trapping manipulates biological molecules by applying extension force or torque to the magnetic beads tethered through biological molecule to pre-coated glass surfaces by manipulating the position or rotation of a pair of magnets. A linear DNA molecule can be manipulated in such a way to generate plectonemes, i.e. DNA supercoils, under an extension force of 0.3 pN (1 pN = 10<sup>–12</sup> N), possessing the feature that the number of unwound base pairs of a supercoiled DNA can be observed by the changes in the number of supercoils reflected by the DNA extension changes. Thus, the DNA unwound by RNAP, i.e. the transcription bubble, during transcription can be observed in this way. By monitoring the kinetics of the transcription bubble in real time, this method thus allows single-molecule detection with single-base resolution and a high-throughput data collection fashion in the kinetic studies of transcription. Owing to the advantages of the manipulation of DNA supercoils with single-molecule magnetic trapping, one can mimic the mechanistic feature of DNAs in vivo and characterize the kinetics of transcription under such conditions. This method can also be combined with single-molecule fluorescence method which can be applied to studying the mechanism of transcription regulation while monitoring the behaviors of fluorescently labeled biological molecules that interact with functional RNAP molecules, providing examples for studying the mechanisms of transcription regulations in more complex systems.

Revealing the DNA Unwinding Activity and Mechanism of Fork Reversal by RecG While Exposed to Variants of Stalled Replication-fork at Single-Molecular Resolution

RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. The elemental mechanism of the helicase activity of RecG with several non-homologous stalled fork structures resembling intermediates formed during the process of DNA repair has been investigated in the present study to capture the dynamic stages of genetic rearrangement. The functional characterization has been exemplified through quantifying the response of the substrate in terms of their molecular heterogeneity and dynamical response by employing single-molecule fluorescence methods. An elevated processivity of RecG is observed for the stalled fork where progression of lagging daughter strand is ahead as compared to that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. RecG is found to adopt an asymmetric mode of locomotion to unwind the lagging daughter strand for facilitating formation of Holliday junction that acts as a suitable intermediate for recombinational repair pathway. Our results emphasize the mechanism adopted by RecG during its ‘sliding back’ mode along the lagging daughter strand to be ‘active translocation and passive unwinding’. This also provide clues as to how this helicase decides and controls the mode of translocation along the DNA to unwind.

Structural basis of early translocation events on the ribosome

Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2−20 per second) and with a low error rate (around 10−3 to 10−5 at each step) over thousands of cycles. The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome.

Direct observation of adsorption and desorption of ds-DNA on graphene oxide and graphene oxide-gold nanoparticle hybrid material: A kineto-mechanistic investigation

Graphene oxide (GO) and its functional derivatives have found tremendous application in the development of biosensors for the detection of nucleic acid hybridization. In this work, we have systematically investigated the adsorption mechanism of oligonucleotides on both GO and graphene oxide-gold nanoparticle (GO-AuNP) hybrid surface and also recovered the adsorbed DNA by applying complementary strands by employing ensemble and single-molecule fluorescence methods. A remarkable observation about the adsorption and desorption mechanism of double-stranded DNA (ds-DNA) on graphene oxide (GO) and GO-AuNP surface has been monitored. The efficiency and rate of adsorption are found to be higher for the GO-AuNP hybrid material compared to that of only GO. The results from the single-molecule-FRET (sm-FRET) indicate that the adsorption of the ds-DNA on the GO-AuNP hybrid material takes place by completely unzipping the strands, i.e. the DNA adsorbs as single-stranded DNA. However, no such clear evidence was observed for the GO. The sm-FRET results reveal the DNA hybridization mechanism to happen “in situ”, i.e. hybridization between the probe and target DNA to form a ds-DNA and desorption thereafter from the GO surface takes place simultaneously. These results also demonstrate that the electrostatic interaction between DNA and GO is of little importance to the overall theory of interaction. This investigation improves the fundamental understanding of DNA hybridization dynamics on GO surface opening new windows in the field of biophysics, sensing, and therapeutic application.

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

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

Structural basis of early translocation events on the ribosome

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

Regulation of E. coli Rep helicase activity by PriC

Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.

Quantification of the Onset of Condensation in Negative Elongation Factors

Several biomolecules condensate in cells to form membraneless organelles via liquid-liquid phase separation, usually in response to changes in their environment. Recent studies suggest that biological phase separation for example regulates cell survival and pathology. The process of biological phase separation itself is not well understood. It is for example unclear how many proteins of a kind are necessary to induce a dense phase in cells – or if the local concentration is the limiting factor. Here we employ highly inclined and laminated optical sheet microscopy and single-molecule fluorescence methods to quantify early steps of phase separation of the negative elongation factor (NELF). NELF downregulates transcription and condensates upon heat and toxin stress. We treat the NELF as a model system to quantify the threshold concentration and rapidness of NELF condensation at low expression levels in fixed and live cells. by counting the number of NELF proteins in the dense phase in a time dependent manner we will contribute to understand the molecular mechanisms underlying liquid-liquid phase separation in cells.

Visualizing Arc mRNA transfer using single molecule fluorescence assays

The transfer of genetic material between cells has been implicated in both maintaining neuronal function as well as the proliferation of many diseases and disorders, especially neurodegenerative disorders. Regulation of mRNA transfer through transport and localization is conducted by RNA binding proteins (RBPs), making RBPs responsible for control of gene expression. The activity‐regulated cytoskeleton‐associated protein, Arc, is an important component of maintaining neuronal synaptic plasticity which ensures the proper flow of cognitive information. In Alzheimer’s disease, an increase of Arc protein expression and localization in synaptic activity areas has been observed in compensating for memory and cognition loss. Although Arc is thought to be released from the synaptic cleft through Arc‐formed capsids to transfer Arc mRNA into new target cells, the mechanism of action is unknown. This project uses single molecule fluorescence methods to interrogate the dynamics of Arc allowing for visualization of the potential movement of Arc capsids/Arc mRNA and the transfer of this genetic information in human cells. Preliminary studies of the Arc using single molecule fluorescence in situ hybridization (smFISH) and live cell imaging techniques has shown a static trajectory of Arc diffusion. Further studies of the transport and transfer of mRNA from Arc capsids will allow for an understanding how this machinery works that can be the basis for the development of new drug delivery methods for diseases with RNA targets.Support or Funding InformationNIH grant number: 1T32GM1320461

Metallation-Induced Heterogeneous Dynamics of DNA Revealed by Single-Molecule FRET.

The metallation of nucleic acids is key to wide-ranging applications, from anticancer medicine to nanomaterials, yet there is a lack of understanding of the molecular-level effects of metallation. Here, we apply single-molecule fluorescence methods to study the reaction of an organo-osmium anticancer complex and DNA. Individual metallated DNA hairpins are characterised using Förster resonance energy transfer (FRET). Although ensemble measurements suggest a simple two-state system, single-molecule experiments reveal an underlying heterogeneity in the oligonucleotide dynamics, attributable to different degrees of metallation of the GC-rich hairpin stem. Metallated hairpins display fast two-state transitions with a two-fold increase in the opening rate to ≈2 s-1 , relative to the unmodified hairpin, and relatively static conformations with long-lived open (and closed) states of 5 to ≥50 s. These studies show that a single-molecule approach can provide new insight into metallation-induced changes in DNA structure and dynamics.

Ratiometric Single-Molecule FRET Measurements to Probe Conformational Subpopulations of Intrinsically Disordered Proteins.

Over the past few decades, numerous examples have demonstrated that intrinsic disorder in proteins lies at the heart of many vital processes, including transcriptional regulation, stress response, cellular signaling, and most recently protein liquid‐liquid phase separation. The so‐called intrinsically disordered proteins (IDPs) involved in these processes have presented a challenge to the classic protein “structure‐function paradigm,” as their functions do not necessarily involve well‐defined structures. Understanding the mechanisms of IDP function is likewise challenging because traditional structure determination methods often fail with such proteins or provide little information about the diverse array of structures that can be related to different functions of a single IDP. Single‐molecule fluorescence methods can overcome this ensemble‐average masking, allowing the resolution of subpopulations and dynamics and thus providing invaluable insights into IDPs and their function. In this protocol, we describe a ratiometric single‐molecule Förster resonance energy transfer (smFRET) routine that permits the investigation of IDP conformational subpopulations and dynamics. We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in‐depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Ratiometric smFRET detection and analysis of IDPs Support Protocol 1: Fluorophore labeling of a protein through maleimide chemistry Support Protocol 2: Sample chamber preparation Support Protocol 3: Determination of direct excitation of acceptor by donor excitation and leakage of donor emission to acceptor emission channel

Relating Structure and Dynamics in RNA Biology.

Recent advances in structural biology methods have enabled a surge in the number of RNA and RNA-protein assembly structures available at atomic or near-atomic resolution. These complexes are often trapped in discrete conformational states that exist along a mechanistic pathway. Single-molecule fluorescence methods provide temporal resolution to elucidate the dynamic mechanisms of processes involving complex RNA and RNA-protein assemblies, but interpretation of such data often requires previous structural knowledge. Here we highlight how single-molecule tools can directly complement structural approaches for two processes--translation and reverse transcription-to provide a dynamic view of molecular function.

Coming Together: RNAs and Proteins Assemble under the Single-Molecule Fluorescence Microscope.

RNAs, across their numerous classes, often work in concert with proteins in RNA-protein complexes (RNPs) to execute critical cellular functions. Ensemble-averaging methods have been instrumental in revealing many important aspects of these RNA-protein interactions, yet are insufficiently sensitive to much of the dynamics at the heart of RNP function. Single-molecule fluorescence microscopy (SMFM) offers complementary, versatile tools to probe RNP conformational and compositional changes in detail. In this review, we first outline the basic principles of SMFM as applied to RNPs, describing key considerations for labeling, imaging, and quantitative analysis. We then sample applications of in vitro and in vivo single-molecule visualization using the case studies of pre-messenger RNA (mRNA) splicing and RNA silencing, respectively. After discussing specific insights single-molecule fluorescence methods have yielded, we briefly review recent developments in the field and highlight areas of anticipated growth.