This work was supported by the National Institutes of Health [R01GM121847]; the Memorial Sloan Kettering Cancer Center (MSKCC) Support Grant/Core Grant (P30 CA008748); and MSKCC Functional Genomics Initiative.

The authors have nothing to disclose.

In comparison to typical in vitro TIRF single-molecule experiments, single-molecule imaging with the assay described here is additionally complex due to the use of cell extract and a high concentration of fluorescently labeled antibody. Compared to the more common practice of one round of surface PEGylation, a second round of PEGylation (step 2) greatly reduces nonspecific antibody binding to detection surface15. The high concentration of diffusing fluorescent antibodies causes an extreme...

Translation in eukaryotic systems occurs predominantly through 7-methylguanosine (m7G) cap-dependent pathways1. Studies indicate that the initiation step of eukaryotic translation is rate-limiting and a common target for regulation2,3,4. Mechanisms of cap-dependent translation have been extensively studied using genetic5, biochemical6,7,8, structural9, and genomic10 bulk approaches. Although these methods have identified diverse mechanisms that regulate cap-dependent initiation, their resolution limits them to ensemble averaging of signals from heterogeneous and asynchronous initiation events. More recently, individual in vivo translation events have been visualized by methods that measure fluorescent antibody binding to epitopes on nascent polypeptides11,12,13,14. However, these new approaches are also limited in their ability to resolve individual initiation events because multiple fluorescent antibodies must bind a nascent peptide to allow single translation events to be resolved from a high intracellular fluorescence background. In many biological interactions, resolved individual kinetic events have provided critical insights into understanding complex multistep and repetitive biological processes that are not possible to synchronize at the molecular level. New methods that can track the dynamics of individual translation events are needed for a better understanding of cap-dependent initiation and regulation.
We recently developed an in vitro assay that measures cap-dependent initiation kinetics with single-molecule resolution15. Considering the large number of known and unknown protein factors involved in this initiation pathway3,16, the single-molecule assay was developed to be compatible with existing in vitro cell-free translation systems to benefit from their preservation of cellular factors and robust translation activity17,18,19,20,21,22,23,24,25. Furthermore, the use of cell-free translation systems allows more compatible comparisons between single-molecule observations and previous bulk results. This approach provides a straight-forward integration of new single-molecule kinetic insights into the existing mechanistic framework of cap-dependent initiation. To establish the single-molecule assay, the traditional cell-free translation system is modified in three ways: an epitope-encoding sequence is inserted at the beginning of the open reading frame (ORF) of a reporter mRNA; the 3&#8242; end of the reporter mRNA is biotinylated to facilitate mRNA end-tethering to single-molecule detection surface; and fluorescently-labeled antibodies are supplemented to the translation extract. These modifications require only basic molecular biology techniques and commonly available reagents. Furthermore, these modifications and the single-molecule imaging conditions preserve the translation kinetics of bulk cell-free translation reactions15.
In this assay (Figure 1), 5&#8242;-end capped and 3&#8242;-end biotinylated reporter mRNA is immobilized to a streptavidin-coated detection surface in a flow chamber. The flow chamber is then filled with a cell-free translation mixture supplemented with fluorescently labeled antibodies. After mRNA translation has occurred for approximately 30-40 codons downstream of the epitope sequence26,27, the epitope emerges from the ribosome exit tunnel and becomes accessible to interact with fluorescently-labeled antibody. This interaction is rapid and its detection by single-molecule fluorescence imaging techniques enables tracking of translation kinetics with single-molecule resolution during active cell-free translation. This assay should broadly benefit in vitro studies of cap-dependent translation kinetics and its regulation, particularly for systems with a working bulk in vitro assay.
A prerequisite for establishing this single-molecule assay is a working bulk cell-free translation assay, which can be achieved using translation extract that is either commercially available or prepared following previously described methods28. Eukaryotic translation extract can be obtained from diverse cells, including fungal, mammalian, and plant28. For imaging, this assay requires a TIRF microscope equipped with tunable laser intensity and incident angle, a motorized sample stage, a motorized fluidics system, and sample temperature control device. Such requirements are generic for modern in vitro single-molecule TIRF experiments and may be achieved differently. The experiment presented here uses an objective-type TIRF system made up of commercially available microscope, software, and accessories all listed in the Table of Materials.

<table>
	<tbody>
		<tr>
			<td>100X oil objective, N.A. 1.49</td>
			<td>Olympus</td>
			<td>UAPON 100XOTIRF</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryamide/bis (40%, 19:1)</td>
			<td>Bio-Rad</td>
			<td>161-0144</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline liquid detergent</td>
			<td>Decon</td>
			<td>5332</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminosilane (N-(2-Aminoethyl)-3-Aminopropyltrimethoxysilane)</td>
			<td>UCT Specialties, LLC</td>
			<td>A0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor ixon Ultra DU 897V EMCCD</td>
			<td>Andor</td>
			<td>DU-897U-CSO-#BV</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor Solis software</td>
			<td>Andor</td>
			<td></td>
			<td>For controlling the Andor EMCCD</td>
		</tr>
		<tr>
			<td>Band-pass filter</td>
			<td>Chroma</td>
			<td>532/640/25</td>
			<td></td>
		</tr>
		<tr>
			<td>Band-pass filter</td>
			<td>Chroma</td>
			<td>NF03-405/488/532/635E-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEG-SVA</td>
			<td>Laysan Bio Inc</td>
			<td>Biotin-PEG-SVA</td>
			<td></td>
		</tr>
		<tr>
			<td>Coenzyme A free acid</td>
			<td>Prolume</td>
			<td>309-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Coolterm software</td>
			<td></td>
			<td></td>
			<td>For controlling the syringe pump</td>
		</tr>
		<tr>
			<td>Desktop computer</td>
			<td>Dell</td>
			<td></td>
			<td>For controlling the microscope, camera, stage, and pump.</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Semrock</td>
			<td>R405/488/532/635</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct-zol RNA microprep 50RNX</td>
			<td>Fisher Scientific</td>
			<td>NC1139450</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Devcon</td>
			<td>14250</td>
			<td></td>
		</tr>
		<tr>
			<td>Firefly luciferin D-Luciferin free acid</td>
			<td>Prolume</td>
			<td>306-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>BP1185500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen perioxide</td>
			<td>Sigma-Aldrich</td>
			<td>216763-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil</td>
			<td>Olympus</td>
			<td>Z-81226A</td>
			<td>Low auto-fluorescence</td>
		</tr>
		<tr>
			<td>Luciferase Assay System</td>
			<td>Promega</td>
			<td>E1500</td>
			<td></td>
		</tr>
		<tr>
			<td>MEGASCRIPT T7 Transcription Kit</td>
			<td>Thermo fisher</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>MMX04751</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Thermo Scientific</td>
			<td>3048</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal anti-FLAG M2-Cy3</td>
			<td>Sigma-Aldrich</td>
			<td>A9594</td>
			<td></td>
		</tr>
		<tr>
			<td>mPEG-SVA</td>
			<td>Laysan Bio Inc</td>
			<td>mPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>MS(PEG)4</td>
			<td>Thermo Scientific</td>
			<td>22341</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (5M)</td>
			<td>Thermo Scientific</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>No 1.5 microscope Cover glass</td>
			<td>Fisherband</td>
			<td>12-544-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Laser, 532nm 100mM</td>
			<td>Olympus digital Laser system</td>
			<td>CMR-LAS 532nm 100mW</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus TirfCtrl software</td>
			<td>Olympus</td>
			<td></td>
			<td>For controlling the laser intensity and incident angle</td>
		</tr>
		<tr>
			<td>Optical table</td>
			<td>TMC vibration control</td>
			<td>63-563</td>
			<td>With vibration isolation</td>
		</tr>
		<tr>
			<td>Phenol chloroform isoamyl alcohol mix</td>
			<td>Sigma-Aldrich</td>
			<td>77617-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce RNA 3&#39; End Biotinylation Kit</td>
			<td>Thermo Scientific</td>
			<td>20160</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide pellets</td>
			<td>Sigma-Aldrich</td>
			<td>P1767-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior motorized XY translation stage</td>
			<td>Prior</td>
			<td>PS3J100</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior PriorTest software</td>
			<td>Prior</td>
			<td></td>
			<td>For controlling the Prior motorized stage</td>
		</tr>
		<tr>
			<td>Recombinant RNasin RNase Inhibitor</td>
			<td>Promega</td>
			<td>N2515</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top Incubator</td>
			<td>In vivo scientific (world precision Instruments)</td>
			<td>98710-1</td>
			<td>With a custom built acrylic cage</td>
		</tr>
		<tr>
			<td>Staining jar</td>
			<td>Fisher Scientific</td>
			<td>08-817</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo Scientific</td>
			<td>43-4301</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Fisher Scientific</td>
			<td>A300212</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR green II</td>
			<td>Fisher Scientific</td>
			<td>S7564</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>1725RN</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard apparatus</td>
			<td>55-3333</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (1M), pH = 7.0</td>
			<td>Thermo Scientific</td>
			<td>AM9850G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Bath</td>
			<td>Branson</td>
			<td>CPX1800H</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5378-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccinia Capping system</td>
			<td>New England Biolabs</td>
			<td>M2080S</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymo-Spin IC Columns</td>
			<td>Zymo Research</td>
			<td>C1004</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro single-molecule imaging assay for the analysis of cap-dependent translation kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

Following the protocol described enables the imaging of individual antibody interactions with nascent N-terminal-tagged polypeptides with single-molecule resolution during active cell-free translation of 3&#39; end-tethered reporter mRNA (Figure 1). A minimal demonstration experiment is reported with the use of three synthetic mRNAs: LUC (encoding untagged luciferase), LUCFLAG (encoding 3xFLAG-tagged luciferase), and hp-LUCFLAG...

Watch this Scientific Journal Video about An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics at JoVE.com

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

an-vitro-single-molecule-imaging-assay-for-analysis-cap-dependent

Research

JoVE Journal

Biochemistry

3.0K Views.  Memorial Sloan Kettering Cancer Center. Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Video: An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...