This research was supported by grant R01NS062842 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health (V.V.D.) and by grants R21 NS064403 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health through ARRA (V.V.D.) and R21 EB006301 National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health (V.V.D.).

The sections for the molecular machine-based detection should be prepared first because their preparation takes more time than the assembly of the molecular device. The construct works well with 5-6&mu;m-thick sections cut from paraformaldehyde-fixed, paraffin-embedded tissue blocks. Use slide brands which retain sections well, such as ProbeOn Plus charged and precleaned slides (Fisher Scientific) or similar. We recommend at first using a tissue with a well-known pattern of DNA damage which contains both DNase I- and DNase II-type breaks, such as dexamethasone-treated apoptotic rat thymus 13,14.
1. Preparation of sections
<ol>
	<li>Place the sections in a slide rack and dewax in xylene for 15 min, transfer to a fresh xylene bath for an additional 5 minutes.</li>
	<li>Rehydrate by passing through graded ethanol concentrations: 96% Ethanol -2x5min; 80% Ethanol - 5min; water - 2x5 min.</li>
	<li>Digest section with Proteinase K. Use 100&mu;L of a 50&mu;g/mL solution per section. Incubate 15' at room temperature (23&deg; C) in a humidified chamber. The time may need adjustment depending on the tissue type. Hard tissues might require longer digestion. Times of 15-25 min are usually used. Insufficient digestion may result in the weaker signal. Overdigestion on the other hand results in signal disappearance and section disruption.</li>
	<li>Rinse in distilled water for 2x10 min.</li>
	<li>Apply 100 &mu;L per section of 2% BSA for preblocking. Incubate for 15 min at room temperature (23&deg; C). During this time assemble the molecular machine.</li>
</ol>
2. Molecular machines assembly
All reagents are scaled for 25 &mu;L total volume, which is sufficient for a single detection in an average size tissue section (10x10mm). The volume can be scaled up as needed.
<ol>
	<li>Combine in a small plastic tube in this order:</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>
			<ol>
				<li>Bidistilled water;</li>
				<li>15% polyethylene glycol-8000;</li>
				<li>Solution of 66 mM-Tris HCl, pH 7.5, 5 mM MgCl2, 0.1 mM dithioerythritol, 1 mM ATP, (i.e. regular T4 DNA ligase buffer);</li>
				<li>70 pmoles of Oligonucleotide 1,</li>
				<li>215 pmoles (1.76 - 7.1 &mu;g) VACC TOPO</li>
				<li>10 units T4 DNA ligase (500 U/mL)</li>
			</ol>
			</td>
		</tr>
	</tbody>
</table>
<ol start="2">
	<li>Mix gently by pipetting. The molecular construct almost instantly self-assembles in this solution and can be used immediately at room temperature (23&deg; C). Increasing the temperature to 37&deg; C blocks the T4 DNA ligase-based component of the labeling.</li>
</ol>
3. Using molecular machines in tissue sections to dual label 5'OH and 5'PO4 DNA breaks
<ol>
	<li>Aspirate the preblocking solution and apply 25&mu;L of the full reaction mix containing 70 pmoles of Oligonucleotide 1, 53 - 215 pmoles (1.76 - 7.1 &mu;g) VACC TOPO and 10 units T4 DNA ligase (500 U/mL) in solution of 66 mM-Tris HCl, pH 7.5, 5mM MgCl2, 0.1 mM dithioerythritol, 1 mM ATP, and 15% polyethylene glycol-8000. The same sequence oligonucleotide carrying a single FITC label, Oligonucleotide 2, can be used instead for detection of a single type of DNA breaks. In such case omit ligase from the labeling reaction. Also in this situation a solution of 50mM Tris-HCL, pH 7.4, 15% PEG-8000 can be used instead of T4 DNA ligase buffer.</li>
	<li>Incubate for 1 hr at room temperature (23&deg; C) in a humidified chamber with a plastic coverslip. Protect from light. Lowering of the temperature to 16&deg; C reduces the ligase-based signal; the temperature increase to 37&deg; C completely eliminates the ligase-based signal. A partial inhibition of ligase sometimes occurs in the reaction mix, possibly due to contaminants introduced with topoisomerase preparations. Therefore, longer incubation (2-4 hours) might be required especially when significant numbers of ligase-labeled breaks are present. Although both ligase and topo signals can be observed at this stage, in many instances the ligase signal can be further enhanced by re-application of the reaction mix without VACC TOPO and dual-labeled Oligonucleotide, but containing a hairpin shaped oligonucleotide (Oligonucleotide 3) (35 mg/mL) and T4 DNA ligase (250 U/mL). To enhance signal proceed to Step 3, to see the reaction without enhancement go to Step 5.</li>
	<li>Remove coverslips by gently immersing the slides vertically in a Coplin jar containing water at room temperature. Aspirate excess water.</li>
	<li>Enhance ligase signal by applying 25 &mu;L of reaction mix without VACC TOPO and Oligonucleotide 1, but containing Oligonucleotide 3: 66 mM-Tris HCl, pH 7.5, 5 mM MgCl2, 0.1 mM dithioerythritol, 1 mM ATP, and 15% polyethylene glycol-8000, 5 units T4 DNA ligase, 35 mg/mL Oligonucleotide 3 (blunt ended hairpin). The total volume of the labeling solution can be scaled up to accommodate the bigger sections. Incubate for 18 hr (overnight) at room temperature (23&deg; C) in a humidified chamber with a plastic coverslip.</li>
	<li>Remove coverslips by gently immersing the slides vertically in Coplin jar containing water at room temperature. Then wash section 3x10 min in distilled water.</li>
	<li>Rinse with sodium bicarbonate buffer. Alkaline solution rinse enhances FITC fluorescence, which is pH sensitive and is significantly reduced below pH 7.</li>
	<li>Cover section with an antifading solution (Vectashield with DAPI), coverslip and analyze the signal using a fluorescent microscope. Double-strand DNA breaks with 5'OH will fluoresce green, 5'PO4 breaks - will fluoresce red (Figure 2).</li>
</ol>
4. Representative Results
<img alt="Figure 2." src="/files/ftp_upload/3628/3628fig2.jpg" /> 
Figure 1. Semi-artificial molecular machine detects two types of DNA damage in situ. The fluorescent machine self-assembles when VACC TOPO binds to the double-hairpin 32-mer. The machine begins operation by splitting itself into two detector units via topoisomerase-made cut at the 3' end of the recognition sequence. This results in a cyclic process where the FITC-labeled unit continuously separates and religates back to the rhodamine-labeled unit. This persists until a detectable DNA break is encountered. When such an alternative acceptor (5'OH blunt end DNA break) is present in the tissue section, the FITC part will ligate to it. The remaining rhodamine part will attach to 5' PO4 DNA blunt end breaks with the help of T4 DNA ligase. Consequently, both types of DNA breaks are simultaneously detected.
<img alt="Figure 1." src="/files/ftp_upload/3628/3628fig1.jpg" />
 
Figure 2. Molecular machine dual labels two types of DNA breaks in a tissue section of dexamethasone-treated thymus. Blunt-ended DNA breaks of DNase I- and DNase II-type are detected in the thymic cortical areas undergoing apoptosis. Green cytoplasmic fluorescence (5'OH DNA breaks) marks cortical macrophages digesting nuclear material of apoptotic thymocytes. This signal is produced by VACC TOPO, and localizes to phagolysosomes with DNA containing 5'OH double-strand breaks 11,12. Red fluorescence (5'PO4 breaks) labels nuclei of apoptotic thymocytes not engulfed by macrophages. Massive numbers of thymocytes simultaneously undergo apoptosis accompanied by generation of 5'PO4 double-strand breaks, visualized by ligase-based labeling. These breaks are located at the nuclear periphery, forming ring-shaped patterns. All cellular nuclei are visualized by counterstaining with DAPI (blue fluorescence).

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No conflicts of interest declared.

In this video, we demonstrate how to assemble and use a dual-labeling DNA damage sensor. The sensor is a molecular machine driven by bio-molecular engine, a virus-encoded protein VACC TOPO linked with artificial components. The presented development exemplifies a bio-enabled approach which advocates adapting biological structures, architectures and actual parts and components of cells to the design of non-toxic molecular scale devices 12,15. This approach resolves two issues inherent to the field of in viv...

in vitro assembly of semi-artificial molecular machine and its use for detection of dna damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

Watch this Scientific Journal Video about In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage at JoVE.com

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

in-vitro-assembly-semi-artificial-molecular-machine-its-use-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.5K Views.  Baylor College of Medicine. We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Video: In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Chemistry

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Biochemistry

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

Genetics

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Precision DNAzyme Nanomachines for Discriminative RNA and DNA Analysis

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

Author Spotlight: Advancements in DNA Nanosensors &#8211; Addressing Sensitivity and Selectivity Challenges in Molecular Detection

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

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 ...

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

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Design and Synthesis of a Reconfigurable DNA Accordion Rack

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

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation