Single-molecule Methods Research Articles

Single-Molecule Characterization of Heterogeneous Soluble Protein Oligomers

Soluble protein oligomers are the primary toxic species in a number of human diseases including Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, cancer, and even certain viral infections. A complicating factor in establishing structure/dynamics/toxicity relationships for soluble oligomers is divergent experimental conditions, such as oligomer preparation protocols, which heavily influence experimental outcomes. Similarly, the low abundance and the structural and dynamic heterogeneity of oligomers are challenges to traditional (ensemble-averaged) biophysical/biochemical characterization. Single-molecule methods are powerful tools for probing this heterogeneity, since oligomers can be observed one at a time. We used single-molecule Förster resonance energy transfer (smFRET) to characterize the heterogeneous structures and dynamics of soluble amyloid-beta (aβ) oligomers prepared using several disparate protocols. The improved comparison between aβ oligomers generated under disparate experimental conditions will better enable correlations to be made between biophysical observables and possible pathways for mediating disease (e.g., direct membrane damage, redox activity, etc.). These correlations are a prerequisite to identifying molecular determinants of oligomer toxicity and therefore means of interfering therapeutically with their mechanisms.

Total Internal Reflection Fluorescence Microscopy and Single-Molecule Kinetics Modules for an Undergraduate Lab Course

The ability to image cellular and molecular phenomena with single-molecule resolution has revolutionized biomedicine and biophysics. Single-molecule methods allow for kinetic analysis of asynchronous processes and for observation of rare events that go undetected in population average measurements. For undergraduate students, single-molecule measurements also offer a direct glimpse into the stochastic nature of events underlying chemical and biophysical processes. We have developed and implemented two modules for teaching advanced microscopy and single-molecule analysis into an undergraduate biophysical chemistry lab course at the University of Colorado Denver.

Single Molecule Experiments Define the Role of Nbs1 and Xrs2 in DNA Tethering by MRN and MRX in DSB Repair

To ensure genome stability and hinder introduction of disease-causing changes, DNA double-strand breaks (DSB) need to be repaired. Homologous recombination (HR) is a key DSB repair pathway. At the start of HR, the MRE11-RAD50-NBS1 (MRN) complex finds the broken ends and upon stimulation of its nuclease activity by CtIP performs the DSB processing for repair. The repair process involves DNA tethering, which is poorly understood. in this study, we use single-molecule analysis to investigate how MRN interacts with long DNA. The method is based on confining long DNA molecules in nanochannels. in contrast to tethering one or both ends to a substrate, as in most common single DNA molecule methods, the DNA is completely free in the nano channels, meaning that reaction on the DNA ends can be studied. Single molecule experiments with MRN and DNA in nanochannels show direct evidence of a strong DNA interaction and tethering of distant DNA fragments by MRN. When comparing MRN and the yeast homologue Mre11-Rad50-Xrs2 (MRX), we observed key similarities. in both systems, the absence of NBS1/Xrs2, lowers the concatemer and circle formation dramatically. Data from single molecule experiments and EMSA show that NBS1 and Xrs2 alone have a strong DNA interaction. These findings strengthen the theory of Nbs1/Xrs2 playing a role in the localization of the MRN/X complex to DNA ends and mediate DNA annealing. Finally, the addition of the MRN/X co-factor CtIP and Sae2, respectively, shows little to no effect on the bridging by MRN/X, although both CtIP and Sae2 exhibit strong DNA bridging on their own. Thus the interaction between MRN/X and the co-factor that is crucial for the nuclease activity is potentially not important for DNA bridging.

Combining Single-Molecule Fluorescence and MD-Simulations to Delineate the Kinetics and Regulation of Proteins

Protein conformational kinetics and its regulation occur on many time- and length-scales. While for example post-translational modifications act very locally, specific protein-protein interaction typically affect larger domains, and global changes (like crowding) affect the whole protein (complex) non-specifically. In a first part we use a combination of single-molecule FRET, FRET-FCS, ns-FCS and MD simulations to delineate the timescales for information transfer from the hydrolysis event in the nucleotide binding site of the heat shock protein 90 (Hsp90) to large conformational changes in the Hsp90 dimer[1]. This allosteric process occurs via a hierarchical mechanism that involves timescales from pico- to microseconds. In a second part we show protein regulation under different degrees of localization, and present the effects on the Hsp90 chaperone system. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We will introduce a new mechanistic concept, namely functional stimulation through conformational confinement[2]. Finally, we use a network of distance distributions obtained with single molecule FRET to investigate a Hsp90 - model client interaction[3]. We find negligible effects of the client on the open-close equilibrium of Hsp90, but strong effects on Hsp90's conformational fluctuations. This hints towards an additional (orthogonal) regulation mechanism. Altogether, these applications demonstrate that a combination of (single-molecule) methods is ideal to investigate the biophysics of multi-component (protein) systems. [1] S. Wolf et al., Allosteric action of nucleotides on Hsp90 across several time- and length scales. BioRxiv, 2020.02.15.950725 (2020); https://doi.org/10.1101/2020.02.15.950725 [2] S. Schmid and T. Hugel, Controlling protein function by fine-tuning conformational flexibility. eLife, 9, 1013 (2020); https://doi.org/10.7554/eLife.57180 [3] Hellenkamp et al., Multidomain structure and correlated dynamics determined by self-consistent FRET networks. Nat Methods, 14 (2), 174 (2017). https://doi.org/10.1038/nmeth.4081

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.

Bayesian Classification and Modeling of Single Molecule Fluorescence Colocalization Images

Multi-wavelength single-molecule fluorescence colocalization methods allow elucidation of complex biochemical reaction mechanisms. However, analysis of colocalization data is an intrinsically challenging and time-consuming problem for multiple reasons. First, to minimize dye photobleaching, images frequently are collected at low signal-to-noise ratios, making it challenging to discriminate real fluorescent spots from noise. Second, transient non-specific interactions of the binder molecule with the surface of the microscope slide can give rise to both false-positive and false-negative detection.

Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Chromatin accessibility profiling methods.

Chromatin accessibility, or the physical access to chromatinized DNA, is a widely studied characteristic of the eukaryotic genome. As active regulatory DNA elements are generally 'accessible', the genome-wide profiling of chromatin accessibility can be used to identify candidate regulatory genomic regions in a tissue or cell type. Multiple biochemical methods have been developed to profile chromatin accessibility, both in bulk and at the single-cell level. Depending on the method, enzymatic cleavage, transposition or DNA methyltransferases are used, followed by high-throughput sequencing, providing a view of genome-wide chromatin accessibility. In this Primer, we discuss these biochemical methods, as well as bioinformatics tools for analysing and interpreting the generated data, and insights into the key regulators underlying developmental, evolutionary and disease processes. We outline standards for data quality, reproducibility and deposition used by the genomics community. Although chromatin accessibility profiling is invaluable to study gene regulation, alone it provides only a partial view of this complex process. Orthogonal assays facilitate the interpretation of accessible regions with respect to enhancer-promoter proximity, functional transcription factor binding and regulatory function. We envision that technological improvements including single-molecule, multi-omics and spatial methods will bring further insight into the secrets of genome regulation.

Lipid Species Dependent Vesicles Clustering Caused by alpha-Synuclein as Revealed by Single-Vesicle Imaging with Total Internal Reflection Fluorescence Microscopy.

Single-molecule methods have been applied to study the mechanisms of many bio-physical systems that occur on the nanometer scale. To probe the dynamics of the such systems including vesicle docking, tethering, fusion, trafficking, protein-membrane interactions, etc., and to obtain reproducible experimental data; proper methodology and framework are crucial. Here, we address this need by developing a protocol for immobilization of vesicles composed of synthetic lipids and measurement using total internal reflection fluorescence (TIRF) microscopy. Furthermore, we demonstrate applications including vesicle clustering mediated by proteins such as alpha-Synuclein (αSyn) and the influence of external ions by using TIRF microscopy. Moreover, we use this method to quantify the dependence of lipid composition and charge on vesicle clustering mediated by αSyn which is based on the methods previously reported.

Single-molecule imaging of pore-forming toxin dynamics in droplet interface bilayers.

Single-channel recording from pore-forming toxins (PFTs) provides a clear and direct molecular readout of toxin action. However to complete any mechanistic understanding of PFT behavior, this functional kinetic readout must be linked to the underlying changes in toxin structure, binding, conformation, or stoichiometry. Here we review how single-molecule imaging methods might be used to further our understanding of PFTs, and provide detailed practical guidance on the use of droplet interface bilayers as a method capable of examining both single-molecule fluorescence and single-channel electrical signals from PFTs.

Evaluation of siRNA Stability and Interaction with Serum Components Using an Agarose Gel-Based Single-Molecule FRET Labeling Method.

Small interfering RNAs (siRNAs) are RNA molecules with promising therapeutic potential as a result of their selective mRNA cleavage. However, despite recent progress, low stability in the bloodstream is an impediment to successful administration in vivo. Thus, the availability of flexible and rapid methods for studying siRNA stability and vehicles is crucial for future novel siRNA-based therapeutics. Herein, we report a fast Förster resonance energy transfer (FRET) method based on agarose gel electrophoresis to evaluate the stability of siRNA in serum as well as siRNA interaction with serum proteins and enzymes.

Bioinformatics Analysis of Cellulose Synthase <i>CesA</i> Gene from <i>Miscanthus lutarioriparius</i>

Based on 3 generations transcriptome sequencing technology-PacBio SMRT single-molecule real-time sequencing method, supplemented with 2 generation sequencing method, a complete and accurate unigene library was constructed. After comparing with closely related species, the cellulose synthase gene ( CesA ) 4 , 7 , 9 gene sequences of Miscanthus lutarioriparius were spliced and named as MlCesA4 , MlCesA7 and MlCesA9 . Then, Bioinformatics analysis software was used to construct phylogenetic trees of proteins, to predict and analyze the phosphorylation sites of post-translational modifications of amino acids, and to predict and analyze conserved domains of proteins.The analysis results showed that the sequence lengths of MlCesA 4, 7, 9 genes were 2980 bp, 3310 bp, and 3208 bp, respectively, which were closely related to Sorghum bicolor and Zea mays ; cellulose synthase of Miscanthus lutarioriparius (MlCesA) MlCesA4 and MlCesA9 have 43 phosphorylation sites, MlCesA7 has 48 phosphorylation sites; MlCesA4 is an unstable protein with 6 transmembrane domains, 4 domains are outside the membrane and 3 are inside the membrane.; MlCesA7 and MlCesA9 are stable proteins, both of which have 8 transmembrane domains, 5 domains are outside the membrane and 4 are inside the membrane. The three are all hydrophilic proteins, and the secondary structure is dominated by α-helix and random coils.

Image processing method for automatic measurement of number of DNA breaks

The number of double-strand breaks can be evaluated from the change of average DNA length. The average DNA length is measured by the single-molecule observation method using fluorescence microscope. The measurement of DNA length in the microscope images is done manually by experienced operators and it is time consuming in many experiments. An image processing method using OpenCV library to measure length of DNA in fluorescence microscope images is developed in this paper. An automation of measurement using deep learning is also proposed.

Real-time imaging of structure and dynamics of transmembrane biomolecules by FRET-induced single-molecule fluorescence attenuation.

Tracking the transmembrane topology and conformational dynamics of membrane proteins is key to understand their functions. It is however challenging to monitor position changes of individual proteins in cell membranes with high sensitivity and high resolution. We review on three single-molecule fluorescence imaging methods - SIFA, LipoFRET and QueenFRET - recently developed in our lab for studying the dynamics of membrane proteins. They can be applied, progressively, to investigate membrane proteins in solid-supported lipid bilayers, artificial liposome membranes and live-cell plasma membranes. The techniques take advantage of the energy transfer from a fluorophore to a cloud of quenchers and are able to extract in real time positions and position changes of a single fluorophore-labeled protein in the direction normal to the membrane surface. The methods have sub-nanometer precision and have proved powerful to investigate biomolecules interacting with bio-membranes.

Imaging the Nanoscale Distribution of Phosphoinositides in the Cell Plasma Membrane with Single-Molecule Localization Super-Resolution Microscopy.

Phosphoinositides make up only a small fraction of cellular phospholipids yet control cell function in a fundamental manner. Through protein interactions, phosphoinositides define cellular organelle identity and regulate protein function and organization and recruitment at the cytosol-membrane interface. As a result, perturbations on phosphoinositide metabolism alter cell physiology and lead to a wide range of human diseases, including cancer and diabetes. Among seven phosphoinositide members, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, also known as PI(4,5)P2 or PIP2) is abundant in the plasma membrane. Besides its role in the second messenger pathway of phospholipase C that cleaves PtdIns(4,5)P2 to form diacylglycerol and inositol-1,4,5-trisphosphate (IP3), PtdIns(4,5)P2 regulates membrane trafficking and the function of the cytoskeleton, ion channels, and transporters. The nanoscale organization of PtdIns(4,5)P2 in the plasma membrane becomes essential to understand cellular signaling specificity in time and space. Here, we describe a single-molecule method to visualize the nanoscale distribution of PtdIns(4,5)P2 in the plasma membrane by using super-resolution microscopy and the dual-color fluorescent probes based on the PLCδ1 pleckstrin homology (PH) domain. This approach can be extended to image other phosphoinositides by changing the specific probes.

Multiplex Single-Molecule Kinetics of Nanopore-Coupled Polymerases.

DNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for specific properties gives the opportunity to identify polymerases with different features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to an α-hemolysin pore on a nanopore array. Here, we use this approach to demonstrate a single-molecule method that enables rapid screening of polymerase variants in a multiplex manner. In this approach, barcoded DNA strands are complexed with polymerase variants and serve as templates for nanopore sequencing. Nanopore sequencing of the barcoded DNA reveals both the barcode identity and kinetic properties of the polymerase variant associated with the cognate barcode, allowing for multiplexed investigation of many polymerase variants in parallel on a single nanopore array. Further, we develop a robust classification algorithm that discriminates kinetic characteristics of the different polymerase mutants. As a proof of concept, we demonstrate the utility of our approach by screening a library of ∼100 polymerases to identify variants for potential applications of biotechnological interest. We anticipate our screening method to be broadly useful for applications that require polymerases with altered physical properties.

Pulsed Interleaved MINFLUX.

We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule localization method with a simplified experimental setup and additional fluorescence lifetime information. In contrast to the original MINFLUX implementation, p-MINFLUX uses interleaved laser pulses to deliver the doughnut-shaped excitation foci at a maximum repetition rate. Using both static and dynamic DNA origami model systems, we demonstrate the performance of p-MINFLUX for single-molecule localization nanoscopy and tracking, respectively. p-MINFLUX delivers 1-2 nm localization precision with 2000-1000 photon counts. In addition, p-MINFLUX gives access to the fluorescence lifetime enabling multiplexing and super-resolved lifetime imaging. p-MINFLUX should help to unlock the full potential of innovative single-molecule localization schemes.

Smart-ORF: a single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames.

Upstream open reading frame (uORF) translation disrupts scanning 43S flux on mRNA and modulates main open reading frame (mORF) translation efficiency. Current tools, however, have limited access to ribosome dynamics in both upstream and main ORFs of an mRNA. Here, we develop a new two-color in vitro fluorescence assay, Smart-ORF, that monitors individual uORF and mORF translation events in real-time with single-molecule resolution. We demonstrate the utility of Smart-ORF by applying it to uORF-encoded arginine attenuator peptide (AAP)-mediated translational regulation. The method enabled quantification of uORF and mORF initiation efficiencies, 80S dwell time, polysome formation, and the correlation between uORF and mORF translation dynamics. Smart-ORF revealed that AAP-mediated 80S stalling in the uORF stimulates the uORF initiation efficiency and promotes clustering of slower uORF-translating ribosomes. This technology provides a new tool that can reveal previously uncharacterized dynamics of uORF-containing mRNA translation.

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics.

SummarySingle-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.

Massively multiplex single-molecule oligonucleosome footprinting.

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure.