We thank K. Chau, J. Morgan, and A. Driller-Colangelo for contributing to the techniques of the segmented DNA origami chassis. We also thank former members of the Reck-Peterson and Shih laboratories for helpful discussions and contributions to the original development of these techniques. We thank J. Wopereis and the Smith College Center for Microscopy and Imaging and L. Bierwert and the Smith College Center for Molecular Biology. We gratefully acknowledge the NSF MRI program for the acquisition of a TIRF microscope.

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The authors have nothing to disclose.

The molecular construction techniques of DNA origami provide a unique way to construct motor ensembles with defined architectures, motor numbers, and types, enabling studies of how emergent behavior arises from specific motor configurations31. As structural and cellular studies continue to elucidate examples of cytoskeletal motors working in teams, techniques for isolating and investigating the biophysical and biochemical mechanisms of motors in ensembles are growing in utility. For example, cryo-...

Dynein and kinesin are cytoskeletal motor proteins responsible for myriad functions in eukaryotic cells1. By converting the chemical energy of ATP hydrolysis into productive work, these motors translocate on microtubules to haul and distribute various intracellular cargos. They also coordinate in the massive intracellular rearrangements associated with mitosis, where they exhibit orchestrated forces that contribute to the positioning and separation of chromosomes. Structural, biochemical, and biophysical assays, including single molecule observations, have revealed the mechanisms of these motors at the individual level (well-reviewed in previous works2,3,4). However, many of the motors&#39; tasks require them to work in small ensembles of both similar and mixed motor types. Comparatively less is understood about the mechanisms that coordinate the activity and ultimate emergent motility of these ensembles5,6. This knowledge gap is due, in part, to the difficulty in creating ensembles with controllable features, such as motor type and copy number. Over the past decade, the molecular construction techniques of DNA origami have been employed to solve this problem. For the microtubule based motors, some examples of these investigations include single molecule observations of ensembles of cytoplasmic dynein-17,8,9, intraflagellar dynein11, various kinesin motors12,13, and mixtures of both dyneins and kinesins7,14,15. Here, we provide details of the purification and oligonucleotide labeling of motors from yeast7,16,17,18,19,20, the folding and purification of segmented DNA origami with tunable compliance8, and the imaging of the yeast motors propelling the chassis structures7,18.
Constructing motor ensembles for in vitro single molecule observation requires three primary efforts. The first is the expression, purification and labeling of motor constructs suitable for attaching to DNA origami. The second is the production and purification of defined DNA origami structures (often termed &#34;chassis&#34;). And the third is the conjugation of the motors to the chassis structure followed by observation using total internal reflection fluorescence (TIRF) microscopy. Here, we provide established protocols for this process for recombinant microtubule-based motors purified from the yeast Saccharomyces cerevisiae7,16,17,18,19. DNA origami-based motor ensembles have been investigated using both recombinant kinesin15 and dynein7,8,18 constructs produced in this yeast expression system16,17,18,19. This protocol is valid for these constructs, given that they are controlled by the galactose induced promoter, and fused to the same protein tags for purification (ZZ and TEV protease linker) and for DNA oligo conjugation (SNAPtag).
Specific yeast strains produce specific motor constructs. For example, the dynein used to study the role of cargo compliance was purified from strain RPY10847,8. In general, strains containing motor constructs with the appropriate genetic modifications for expression and purification can be requested from the laboratories having published the use of those motors. Constructs with novel attributes such as mutations or tags can be made using recombinant genetic techniques, such as lithium acetate transformation21 and commercial kits. Detailed protocols for creating modified motor proteins in yeast for single molecule studies have been published19. In addition to the motors being fused to the SNAPtag, the oligos used to label the motors must be conjugated to the SNAP substrate, benzylguanine (BG); previously published protocols describe the formation and purification of BG-oligo conjugates18. The overall strategy described here has also been employed for actin-based motors (see previous works for examples22,23,24), and motors purified from other organisms and expression systems (see previous works for examples7,9,10,11,12,13,14).
Polymerized microtubules (MTs) are used in these experiments in two different procedures. MT affinity purification of functional motors requires MTs that are not labelled with other functional groups, while the motor-ensemble motility TIRF assay requires MTs labeled with biotin and fluorophores. In all cases, MTs are stabilized with taxol to prevent denaturation. The MT affinity purification step is used to remove any non-motile motors with a high MT affinity, as these motors can alter ensemble motility if conjugated to a chassis. During this process, active motors unbind the MTs and remain in solution, while tight-binding motors spin down in the MT pellet. This helps ensure all motors on the chassis are from an active population.
A variety of DNA origami structures have been used to study cytoskeletal motor ensembles. As the mechanistic understanding of ensemble transport has increased, the DNA origami structures employed in experiments have grown in complexity. In principle, any structure could be adapted for this purpose provided it is modified to include single-stranded DNA attachment sites for motors and fluorophores. Specific chassis designs and attributes may be useful for probing particular questions about the emergent behavior of motors ensembles. For example, rigid rods have been used to develop foundational knowledge of how copy number affects transport by teams of dyneins and kinesins7,15,18, and 2D platforms have been used to study myosin ensemble navigation of actin networks22. Structures with variable or tunable flexibility have been used to understand the roles of elastic coupling between motors and to probe how stepping synchronization affects motility8,24. More recently, spherical structures are being used to gain insight into how geometrical constraints to motor-track binding affect the dynamics of motility25.
In this protocol, we offer specific steps for ensemble experiments on segmented chassis with variable rigidity. Binding sites on the chassis are sometimes referred to as &#34;handles&#34;, while complementary DNA sequences that bind these handles are termed &#34;antihandles&#34;. The number of motors on these chassis is determined by which segments contain extended handle staples with complementarity to the antihandle oligo on the oligo-labeled motors. Using different handle sequences on different segments allows for binding of different types of motors to specific locations on the chassis. The chassis detailed here is composed of 7 sequential rigid segments, each comprised of 12 double-stranded DNA helices arranged in 2 concentric rings8. The rigid segments contain the motor handles and are connected through regions that can be either flexible single-stranded DNA or rigid double-stranded DNA, depending on the absence or presence, respectively, of &#34;linker&#34; staples. The compliance of the chassis structure is thus determined by the presence or absence of these &#34;linker&#34; staples. See previous reports for further details and specific DNA sequences8. In addition, multiple methods can be used to purify chassis26. The rate-zonal glycerol gradient centrifugation method27 is described here.

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			<td>2 mL Round Bottom Tube</td>
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			<td>2 mL at 400nM</td>
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			<td>Promega</td>
			<td>V6101</td>
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			<td>Invitrogen</td>
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			<td>Tubulin protein (&#62;99% pure): porcine brain</td>
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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.

Successful purifications of motors and chassis structures were assayed by gel electrophoresis. SDS-PAGE analysis confirmed the successful extraction of dynein from yeast (Figure 2), as the final filtrate collected in step 2.3.7 showed a clear, sharp band at the position of ~350 kDa. As expected, this dynein band was absent from the flowthrough and wash that removed unwanted proteins, and the beads from which dynein was cleaved. The observation suggests that t...

Watch this Scientific Journal Video about Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation at JoVE.com

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

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 ...

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6.5K Views.  Smith College. 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.

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

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 ...

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 ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

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 ...

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 ...