Name: single-molecule imaging of ews-fli1 condensates assembling on dna
Uploaded: 2021-09-08T15:00:00
Description: Watch this Scientific Journal Video about Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA at JoVE.com

This work was supported by NSFC Grants No. 31670762 (Z.Q.).

1. Preparation of the lipid bilayer master mix
<ol>
	<li>Rinse glass vials with double-distilled water (ddH2O) and 99% ethanol and dry them in a 60 &#176;C drying oven.</li>
	<li>Make the lipid master mix by dissolving 1 g of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 100 mg of polyethylene glycol-reacted (PEGylated) lipids (18:1 of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (PEG2000 DOPE) and 25 mg of biotinylated lipids (18:1 of 1,2-dioleoyl-s...

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As single-molecule approaches are extremely sensitive to the contents of the reaction system, extra effort must be invested to ensure good quality of all the materials and solutions during the DNA Curtains experiments, especially the lipids prepared in sections 1 and 2 and the buffers used in section 5. Reagents of higher purity must be used to prepare buffers, and buffers must be freshly prepared for the single-molecule assay
When 500 nM mCherry-labeled EWS-FLI1 was flushed into the chamber, ...

Transcriptional regulation is a crucial step for precise gene expression in living cells. Many factors, such as chromosomal modification, transcription factors (TFs), and non-coding RNAs, participate in this complicated process1,2,3. Among these factors, TFs contribute to the specificity of transcriptional regulation by recognizing and binding to specific DNA sequences known as promoters or enhancers and subsequently recruiting other functional proteins to activate or repress transcription4,5,6,7. How these TFs manage to search for their target sites in the human genome and interact with DNA coated with histones and non-histone DNA-binding proteins has perplexed scientists for decades. In the past few years, several classical models for the target search mechanism of TFs have been built to describe how they &#34;slide,&#34; &#34;hop,&#34; &#34;jump,&#34; or &#34;intersegment transfer&#34; along the DNA chain8,9,10,11. These models are focused on the searching behavior on the DNA of one single TF molecule. However, recent studies show that some TFs undergo liquid-liquid phase separation (LLPS) either alone in the nucleus or with the Mediator complex12. The observed droplets of TFs are associated with the promoter or enhancer regions, highlighting the role of biomolecular condensate formation in transcription and the three-dimensional genome13,14,15. These biomolecular condensates are linked to membrane-lacking compartments in vivo and in vitro. They are formed via LLPS, in which modular biomacromolecules and intrinsically disordered regions (IDRs) of proteins are two main driving forces of multivalent interactions16. Thus, TFs not only search DNA but also function synergistically within these condensates4,17,18. To date, the biophysical property of these transcription condensates on DNA remains unclear.
Therefore, this study aimed to apply a single-molecule method-DNA Curtains-to directly image the formation and dynamics of the transcription condensates formed by TFs on DNA in vitro. DNA Curtains, a high-throughput in vitro imaging platform to study the interaction between proteins and DNA, has been applied in DNA repair19,20,21, target search22, and LLPS17,23,24. The flowcell of DNA Curtains is coated with biotinylated lipid bilayers to passivate the surface and allow the biomolecules to diffuse on the surface. The nanofabricated zig-zag patterns limit the movement of DNA. Biotinylated Lambda DNA substrates can align along the barrier edges and be stretched by the oriented buffer flow. The same starting and ending sequences of all the molecules allow the tracking of the protein on DNA and describe the position distribution of the binding events25,26. Moreover, the combination of DNA Curtains with total internal reflection fluorescence microscopy (TIRFM) helps minimize the background noise and detect signals at a single-molecule level. Thus, DNA Curtains could be a promising method to investigate the dynamics of transcription condensate formation on DNA motifs. This paper describes the example of an FUS/EWS/TAF15 (FET) family fusion oncoprotein, EWS-FLI1, generated by chromosomal translocation. Lambda DNA containing 25&#215; GGAA-the binding sequence of EWS-FLI127- was used as the DNA substrate in the DNA Curtains experiments to observe how EWS-FLI1 molecules undergo LLPS on DNA. This manuscript discusses the experimental protocol and data analysis methods in detail.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>488 nm diodepumped solid-state laser</td>
			<td>Coherent</td>
			<td>OBIS488LS</td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm diodepumped solid-state laser</td>
			<td>Coherent</td>
			<td>OBIS561LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Rhawn</td>
			<td>R003215-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated DOPE</td>
			<td>Avanti</td>
			<td>870273P</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Amresco</td>
			<td>1595C027</td>
			<td></td>
		</tr>
		<tr>
			<td>Coating Electra 92</td>
			<td>Allresist GmbH</td>
			<td>AR-PC 5090.02</td>
			<td>The conductive protective coating</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I bovine</td>
			<td>Sigma</td>
			<td>D5139-2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti</td>
			<td>850375P</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Hellmanex III</td>
			<td>Sigma</td>
			<td>Z805939-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>60130</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda DNA</td>
			<td>NEB</td>
			<td>N3013S</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda Packing Extracts</td>
			<td>Epicentre</td>
			<td>MP5120</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>s3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoport</td>
			<td>Idex</td>
			<td>N-333-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NheI-HF</td>
			<td>NEB</td>
			<td>R3131S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Inverted Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>NZCYM Broth</td>
			<td>Sigma</td>
			<td>N3643-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-2000 DOPE</td>
			<td>Avanti</td>
			<td>880130P-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-8000</td>
			<td>Amresco</td>
			<td>25322-68-3</td>
			<td></td>
		</tr>
		<tr>
			<td>PMMA 200K, ETHYL LACTATE 4%</td>
			<td>Allresist GmbH</td>
			<td>AR-P 649.04</td>
			<td></td>
		</tr>
		<tr>
			<td>PMMA 950K, ANISOLE 2%</td>
			<td>Allresist GmbH</td>
			<td>AR-P 672.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Prime 95B Scientific CMOS camera</td>
			<td>PHOTOMETRICS</td>
			<td>Prime95B</td>
			<td></td>
		</tr>
		<tr>
			<td>proteinase K</td>
			<td>NEB</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica glass slide</td>
			<td>G.Finkenbeiner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Six-way injection valve</td>
			<td>Idex</td>
			<td>MXP9900-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo</td>
			<td>S888</td>
			<td>Diluted with ddH2O</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>Pump11 Elite</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>NEB</td>
			<td>R0146V</td>
			<td></td>
		</tr>
		<tr>
			<td>YOYO-1 Iodide (491/509)</td>
			<td>Invitrogen</td>
			<td>Y3601</td>
			<td>Diluted with DMSO</td>
		</tr>
	</tbody>
</table>

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.

The schematic of DNA Curtains is shown in Figure 1A, Figure 1B, and Figure 1D. The cloned target sequence containing 25 uninterrupted repeats of GGAA is found in the NORB1 promoter in Ewing sarcoma. This target sequence is crucial for EWS-FLI1 recruitment28. EWS-FLI1 molecules were visualized by detecting the mCherry-labeled EWS-FLI1 signals obtained with a 561 nm laser (Figure 1...

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

We provide a single molecule assay to observe the phase separation of transcription factors on DNA This technique allows realtime realization of the dynamic process of transcription factors assembling as a liquid droplet on DNA. EWS-FL1 is one of the most frequently involved fusion genes in Ewing sarcoma tumorigenesis. Our research could identify the relationship between DNA motif number and EWS-FL1 condensate formation which is associated with a barren gene transcription in tumor cells, and maybe helpful for the prognosis.This method could be applied to study any transcription condensates or complexes in vitro, like P53 and MED1. To prepare a flow cell with zigzag nano fabricated barriers for a DNA curtain experiment, connect the input and output tubes in the correct direction. Then wash the flow cell with 2.5 milliliters of lipid buffer using two three milliliter syringes, ensuring there is no bubble in the flow cell.Remove the syringe from the output and inject one milliliter of the liposome solution three times with a five minute incubation between each injection. For healing, rewash the flow cell with 2.5 milliliters of lipid buffer slowly, and incubate at room temperature for 30 minutes. During the incubation, prepare the BSA buffer as mentioned in the text manuscript.Then after 30 minutes of healing, wash the flow cell with 2.5 milliliters of BSA buffer from the output side. Next, inject 800 microliters of streptavidin buffer into the flow cell from the input side in two steps with a 10 minute incubation after each step. Then wash the flow cell with 2.5 milliliters of BSA buffer to remove all free streptavidin molecules.Next, dilute the biotin Lambda DNA with cloned motifs using BSA buffer and inject it four times slowly at five minute intervals. During the injection, turn on the microscope in the scientific complimentary metal oxide semiconductor system. Then wash the tubing with 10 milliliters of double distilled water and rinse the prism and the tubing connector with water, 2%liquid cuvette cleaner, and 99%ethanol.Next, draw up at least 20 milliliters of the imaging buffer into a 30 milliliter syringe. Set up the flow cell on the microscope stage and connect it to the microfluidic system. Using a flow rate of 0.03 milliliters per minute, flush the DNA molecules to the barrier for 10 minutes.Then switch off the microfluidic system and incubate for 30 minutes. with the flow stopped, allowing the DNA to diffuse laterally. To image the EWS-FLI1 condensation formation on DNA curtains, open the imaging software and find and mark the positions of the nine zigzag patterns under bright-field.Then turn on the flow at 0.2 milliliters per minute to stain the DNA with double stranded DNA dye for 10 minutes. Next, dilute the mCherry EWS-FLI1 protein with the imaging buffer at a concentration of 100 nanomoles in 100 microliters. Then load the protein sample through the valve with a 100 microliter glass syringe and change the flow rate to 0.4 milliliters per minute.After turning on the 488 nanometer laser pre-scan each region to check the DNA distribution state and select the region in which the DNA molecules distribute evenly. Then set the laser power to 10%for the 488 nanometer laser and 20%for the 561 nanometer laser. And using the power meter, measure the real laser power near the prism.Start acquiring images at two second intervals with both 488 and 561 nanometer lasers simultaneously. Then change the valve from the manual mode to injection mode to let the imaging buffer flush the protein sample into the flow cell after 60 seconds. To remove the free EWS-FLI1 keep washing the flow cell with the imaging buffer for five minutes with only the 561 nanometer laser switched on.Then stop the flow and incubate at 37 degrees Celsius for 10 minutes. After 10 minutes, turn on the flow at 0.4 milliliters per minute to let the DNA extend and acquire images at two second intervals between different frames to obtain high throughput data of EWS-FLI1 condensate formation. EWS-FLI1 molecules were visualized by detecting the mCherry labeled EWS-FLI1 signals obtained with a 561 nanometer laser.The in vitro formation of EWS-FLI1 condensate at the site of the 25 GGAA repeats in the DNA substrate could be directly visualized. The specificity of the mCherry EWS-FLI1 used in DNA curtains was confirmed by an electrophoretic mobility shift assay using a DNA template with and without the 25 GGAA repeats separately. When the EWS-FLI1 concentration was titrated from 20 to 500 nanomoles the EWS-FLI1 intensity increased dramatically.Whereas the change in the FLI one DBD intensity was negligible when the proteins were saturated to cover the GGAA repeats, suggesting that EWS-FLI1 formed condensates on DNA. The injection should be very slow and soft so that the liposome and DNA could distribute evenly on the flow cell. It is important to perform MSA to confirm that a purified transcription factor specifically bonds with target sequence in vitro.This technique allows people to explore whether the condensate will facilitate the target search of transcription factors, and its effect on transcription.

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Biochemistry

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 ...

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

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 ...

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 ...

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.

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.

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

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 Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...