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...

Watch this Scientific Journal Video about Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA at JoVE.com

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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Research

JoVE Journal

Biochemistry

2.2K ビュー.  Peking University. DNA上の転写因子の相分離を観察するための単一分子アッセイを提供します この技術により、転写因子がDNA上に液滴として集合する動的なプロセスをリアルタイムで実現できます。EWS-FL1は、ユーイング肉腫腫瘍形成において最も頻繁に関与する融合遺伝子の1つです。私たちの研究は、DNAモチーフ数と、腫瘍細胞における不毛な遺伝子転写に関連するEWS-FL1凝縮物形成との?...

ビデオ: Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

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, ...

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-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 Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Imaging the Motion of Fully Reconstituted Cdc45/Mcm2-7/GINS (CMG)

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical trap.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication ...

Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper ...

In Situ Nucleosome Reconstitution for Single-Molecule Studies

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

Ensemble Assembly and Activation of Fluorescently Labeled CMG onto Magnetic Bead-Bound DNA for Single Molecule Imaging

To begin, take one 75 microliter aliquot containing one milligram of DNA-bound streptavidin-coated magnetic beads and remove the supernatant with the magnetic rack. Wash the beads with 200 microliters of loading buffer. Then resuspend the beads in 75 microliters of loading buffer and mix gently by pipetting.Next, add the origin recognition complex at a final concentration of 37.5 nanomolar to the bead-bound DNA and incubate the reaction for five minutes at 30 degrees Celsius with 800 revolutions per minute agitation. Add CDC6 at a final concentration of 50 nanomolar and incubate the reaction as demonstrated before. Then add Mcm2-7 Cdt1 at a final concentration of 100 nanomolar and incubate the reaction for 20 minutes as demonstrated previously.After that, add DDK at a final concentration of 100 nanomolar and incubate similarly for 30 minutes. After removing the supernatant, wash the bead-bound DNA containing phosphorylated Mcm-2-7 hexamers with 200 microliters of high salt wash buffer. Mix by pipetting, ensuring the beads are fully resuspended.Next, remove the buffer and wash the beads once with 200 microliters of CMG buffer. To assemble and activate the fluorescently-labeled CMG onto bead-bound DNA, remove the CMG buffer and resuspend the DNA-bound beads in 50 microliters of CMG buffer supplemented with five millimolar ADP. Next, mix all the components mentioned on the screen in one tube and place it on ice.Immediately add the resuspended bead-bound DNA to the protein mix. Incubate the reaction for 15 minutes at 30 degrees Celsius with 800 RPM agitation. Wash the bead sequentially with HSW buffer and CMG buffer.After removing the buffer, resuspend the CMG containing DNA-bound magnetic beads in 200 microliters of elution buffer, then incubate at room temperature for one hour with 800 RPM agitation. Place the tube in a magnetic rack and allow the beads to be collected for five minutes. Carefully collect the supernatant containing the eluted DNA-CMG complexes without disrupting the settled beads and transfer it to a new tube.To ensure that no beads remain in the solution, place the collected supernatant again in a magnetic rack and keep for another five minutes carefully. Collect the supernatant and transfer it to a new tube. Add 1, 400 microliters of CMG buffer to the 200 microliters of supernatant.The sample is now ready for single molecule imaging.