This work was supported by the National Science Foundation grant MCB-1715317.

1. Making the flow cell
<ol>
	<li>Washing the coverslips
	<ol>
		<li>Place coverslips in staining jars and sonicate with ethanol (EtOH), then with 1 M KOH (for 30 min each). To avoid KOH precipitation in EtOH, rinse thoroughly with ddH2O between washes.</li>
		<li>Repeat both EtOH and the KOH washing steps 1x for a total number of four washes (two EtOH and two KOH). Store cleaned coverslips in ddH2O in staining jars.</li>
	</ol>
	</li>
	<li>Cut loading and exit tubes (8 cm long) using a clean razor and insert into holes in a clean glass slide. Use PE-20 for inlet and PE-60 for outlet. Epoxy for 5 min to secure tubing and trim off any excess tubing. 
	NOTE: Glass slides measure 2&#39;&#39; x 3&#39;&#39; x 1 mm. Holes are drilled in pairs 15 mm apart. Each pair forms either end of a single channel. The tubing with the smaller ID is used for the inlet, as this reduces the upstream dead volume and thus the mixing time required (Figure 1).</li>
	<li>Line up and apply precut double-sided tape with channel pattern cut out over holes on the glass slide. Smooth out with plastic forceps to achieve a good seal. 
	NOTE: The double-sided tape used in this experiment is 120 &#181;m thick, and the channels are 2 mm wide and 15 mm long. Channels are cut using a knife printer (Table of Materials). It is possible to fit up to four channels on a single coverslip. See Figure 1 for an image of the flow cell.</li>
	<li>After peeling off the backing, apply a clean coverslip (dried with compressed air) over the tape and smooth out again with plastic forceps for a good seal.</li>
	<li>Epoxy the edge off the coverslip to seal the flow cell and let it cure.</li>
</ol>
2. Preparation of labeled DNA for tethering
<ol>
	<li>In a PCR tube, prepare 50 &#181;L of PCR reaction mix containing 0.02 U/&#181;L high fidelity DNA polymerase, 200 &#181;M dNTPs, 0.5 &#181;M forward primer, 0.5 &#181;M reverse primer, and 250 ng of M13mp18 vector DNA. 
	NOTE: Here, the forward primer (biotin-CCAACTTAATCGCCTTGC) and reverse primer (digoxigenin-TGACCATTAGATACATTTCGC) were chosen to amplify a region approximately 1,000 bp long, spanning from positions 6338 to 107 in the circular genome of the M13mp18 DNA. There is a single NdeI site in the middle of the amplified region. The forward primer is 5&#697; labeled with digoxigenin, which binds the anti-digoxigenin on the coverslip. The reverse primer is 5&#697; labeled with biotin, which binds the streptavidin-coated beads.</li>
	<li>Insert the PCR tube in thermocycler and follow the cycle as shown in Table 1.</li>
	<li>Purify the PCR product with a PCR clean-up kit following the manufacturer&#39;s protocol. 
	NOTE: Using the kit specified in the Table of Materials, the typical DNA yield is ~2 &#181;g.</li>
</ol>
3. Tethering of DNA and beads
<ol>
	<li>Prepare 10 mL of buffer A (1 M Tris-HCl [pH = 7.5], 50 mM NaCl, 2 mM MgCl2, 1 mg/mL &#946;-Casein, 1 mg/mL Pluronic F-127). Degas in a vacuum desiccator for at least 1 h.</li>
	<li>To functionalize flow cell, inject 25 &#181;L of anti-digoxigenin FAB fragments (20 &#181;g/mL) in PBS into the flow channel. Use gel loading tips to fit into the PE-60 tubing. Incubate at room temperature (RT) for 30 min.</li>
	<li>After incubation, flush the channel by pulling 0.5 mL of buffer A through the channel using a syringe. Take care not to introduce air into the channel.</li>
	<li>After functionalization, mount the flow cell on an inverted microscope. Hook up the outlet tube to a syringe pump and put the inlet tube into a microcentrifuge tube containing buffer A.</li>
	<li>Manually pull at least 0.5 mL of buffer A to flush the system and prime the pump. Let the pump run at 10 &#181;L/min for at least 5 min to equilibrate the system.</li>
	<li>To prepare the beads (Table of Materials), vortex the stock bottle of beads and pipette 1.6 &#181;L of the 10 mg/mL stock beads into 50 &#181;L of buffer A, then vortex.</li>
	<li>Using a magnetic separator, pipette out the buffer and resuspend in 50 &#181;L of buffer A, then vortex.</li>
	<li>Repeat step 3.7 2x for a total of three washes. For the last wash, resuspend in 100 &#181;L of buffer A and vortex to achieve a final concentration of 160 &#181;g/mL.</li>
	<li>To complex the DNA and beads, first prepare 480 &#181;L of 0.5 pM labeled DNA substrate in buffer A. Then, pipette in 20 &#181;L of 160 &#181;g/mL bead suspension, making sure to vortex the beads before pipetting. Place on a rotator for 3 min.</li>
	<li>After 3 min, immediately load into channel at a flow rate of 10 &#181;L/min for ~15 min or until sufficient bead tethering is observed. 
	NOTE: Beads should not be so densely packed that they interact with each other on the surface (see discussion section).</li>
	<li>To wash the channel of all free beads, switch the inlet tube to a fresh tube of buffer A and flow in at 50 &#181;L/min for at least 10 min or until no loose beads are observed.</li>
</ol>
4. Data collection and analysis
<ol>
	<li>To prepare for data collection, place inlet tube into a microcentrifuge tube containing at least 100 &#181;L of NdeI (0.25&#8211;4.00 U/mL) in buffer A. Lower the permanent magnet over the flow channel, and position the light source off axis for darkfield imaging. 
	NOTE: Two annular rare earth magnets, epoxied together, are held 8 mm above the active surface of the flow channel using a cantilevered optical post during data collection. A goose neck lamp is used for the off-axis light course.</li>
	<li>Use a commercial microscope, video camera, and data collection software (Table of Materials). In the software, click the &#34;Exposure&#34; tab, and set &#34;Exposure Time&#34; to 10 ms. Click &#34;Timelapse&#34; tab and set &#34;Image Count&#34; to 600, &#34;Duration&#34; to 20 min, and &#34;Interval&#34; to 2 s. Click &#34;Run&#34; to start data collection.</li>
	<li>On a syringe pump, set the flow rate to 150 &#181;L/min and injection volume to 80 &#181;L. Press &#34;Run&#34; at 1 min into data collection. After injection, turn off the pump and close the valve to prevent flow during data collection.</li>
	<li>Once data is collected, open image analysis software (Table of Materials). Under the &#34;File&#34; tab, choose &#34;Import&#34; | &#34;Image Sequence&#34;. Locate the image files in the pop-up menu and click &#34;Open&#34;.</li>
	<li>Set the threshold by choosing &#34;Adjust Theshold&#34; under the &#34;Image&#34; pull-down menu. Use the slider bar to set the threshold value to identify bright spots corresponding to beads in the image.</li>
	<li>Count the bright spots in each frame by clicking &#34;Analyze Particles&#34; in the &#34;Analyze&#34; pull-down menu. Click &#34;OK&#34;, then choose &#34;Yes&#34; to process all images. Save the results file. 
	NOTE: This will save a data file that contains the number of beads in each recorded video frame.</li>
	<li>Open data analysis software (Table of Materials) and import the results file by clicking &#34;Import from Text File&#34; in the &#34;File&#34; pull-down menu. Plot the bead count data vs. time.</li>
	<li>Fit the number of beads vs. time by clicking &#34;Curve Fit&#34; in the &#34;Analyze&#34; pull-down menu. Choose the &#34;Natural Exponent&#34; equation and click &#34;Try Fit&#34; | &#34;OK&#34;. 
	NOTE: Only the data recorded after the injection of sample should be included in the fitting region. The fit parameter in the exponent of the fitting function will be the cleavage rate.</li>
</ol>

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The authors have no conflicts of interest to disclose.

The protocol can be used to measure the kinetics of any SSDC system, provided that the strand separation is observed during the experiment. The detection of cleavage is affected by observing the detachment of the tethered bead and therefore marks the instant of strand separation. All preceding steps occur before the detection of the cleavage; thus, only the total transit time is recorded.
The flow cell coverslip is functionalized via non-specific adsorption of antibody protein to the clean gla...

Site-specific DNA cleavage (SSDC) is a key step in many genomic transactions. For example, bacterial restriction-modification (RM)1 and CRISPR2 systems protect cells from attack by phages and plasmids by recognizing and cleaving foreign DNA at specific sequences. In type II RM, restriction endonucleases (REs) recognize short 4&#8211;8 base pair (bp) sequences via protein-nucleic acid interactions3. CRISPR-associated endonucleases, such as Cas9, bind to sites via hybridization of the target site with crRNAs bound to the endonucleases4. The creation of site-specific double stranded breaks (DSBs) are also the first step in many DNA recombination events5. For example, the diversity of antigen binding regions created by V(D)J recombination requires the recognition and cleavage of specific target sites6. Some transposons are known to target specific DNA sequences, as well7. Not surprisingly, many site-specific nucleases involved in these processes, such as Cas9, are a key component of gene editing technologies8. In addition, novel site-specific endonucleases (i.e., zinc finger nucleases9 and TALENS10) have also been engineered to edit genomes.
Many methods have been employed to measure the kinetics of site-specific cleavage of nucleic acids. These include gel analysis, fluorescence11,12, and sequencing based methods13. A major advancement was achieved with the tethering of microbeads, which allows DSBs in single molecules of DNA to be detected by the motion of a bead after strand separation. In these methods, different types of forces are employed to ensure strand separation and motion of the bead post-cleavage. In one case, optical traps have been used to measure cleavage of DNA by EcoRV14. In these experiments, target search is the objective of the investigation, with conditions optimized so that site-specific binding is the rate limiting step. One drawback of optical traps is that only a single DNA can be observed at a time. In addition, a periodic large pulling force has to be applied to test for the strand separation.
Another technique uses a combination of flow and weak magnetic forces to pull on the bead in a continuous manner15. In this way, diffusion limited cleavage by NdeI is measured. The method employed allows for the simultaneous measurement of several hundred DNAs at once, allowing for statistical significance to be attained in a single experiment. Experiments based on magnetic tweezers have also been used. In one such study, a retroviral integrase was studied by including a DSB in the insertion oligonucleotide16. Successful integration resulted in the incorporation of DSB in the tethered DNA and loss of the attached bead. In a similar study of the ATP-dependent type III restriction endonuclease EcoPI, tens of DNAs were observed in a single experiment17. Magnetic tweezers hold the advantage that tension, as well as DNA looping, can be controlled and monitored during the reaction.
Presented here is a highly parallel single molecule method for measuring SSDC kinetics, which takes advantage of recent improvements in large-scale tethering of DNAs. This method is an improvement and extension of previous methods used to measure DNA replication18, contour length of DNA19, and cleavage by REs15. In this technique, linear DNAs containing a single copy of the recognition sequence are prepared with biotin at one end and digoxigenin at the other. The biotin binds streptavidin, which is covalently attached to a paramagnetic microbead. The DNA-bead complexes are injected into a microfluidic channel that has been functionalized with anti-digoxigenin FAB fragments. The DNA then tethers to the surface attachment points via binding of the digoxigenin to the adsorbed FAB fragments. Weak magnetic forces applied with a permanent magnet keep the bead from sticking non-specifically to the surface. Samples can be injected rapidly (&#60;30 s) into the flow channel to activate the cleavage reaction. Flow is turned off during data collection. As each DNA is cleaved, the exact time of cleavage can be determined by recording the frame in which the bead moves up and out of the focal plan of the objective, thus disappearing from the video record. A frame-by-frame count of remaining beads can be used to quantify the reaction progress.
Presented below is the complete protocol as well as example data collected using NdeI. As an example of how the technique can be applied, cleavage rates for a range of protein concentrations are measured at two different concentrations of magnesium, an essential metal cofactor. Although this application of the protocol uses NdeI, the method can be adapted for use with any site-specific nuclease by varying the DNA substrate design.

<table><tbody><tr><td>5 minute Epoxy</td><td>Devcon</td><td>14250</td><td></td></tr><tr><td>anti-digoxigenin FAB fragments</td><td>Roche Diagnostics</td><td>11214667001</td><td></td></tr><tr><td>camera and software</td><td>Jenoptik</td><td>GRYPHAX SUBRA</td><td></td></tr><tr><td>data analysis software</td><td>Vernier Inc.</td><td>LP</td><td></td></tr><tr><td>double sided tape</td><td>Grace Biolabs</td><td>SA-S-1L</td><td></td></tr><tr><td>Dulbeccos Phosphate Buffered Saline</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>ethanol 95%</td><td>VWR</td><td>89370-082</td><td></td></tr><tr><td>forward primer: digoxigenin-CCAACTTAATCGCCTTGC</td><td>Integrated DNA Technologies</td><td>n/a</td><td></td></tr><tr><td>image analysis software</td><td>National Institutes of Health</td><td>ImageJ</td><td></td></tr><tr><td>inverted microscope</td><td>Nikon</td><td>TE2000</td><td></td></tr><tr><td>knife printer</td><td>Silhouette</td><td></td><td></td></tr><tr><td>M13mp18 DNA</td><td>New England Biolabs</td><td>N4040S</td><td></td></tr><tr><td>MyOne streptavidin beads</td><td>Thermo Fisher Scientific</td><td>65601</td><td></td></tr><tr><td>NdeI enzyme</td><td>New England Biolabs</td><td>R0111S</td><td></td></tr><tr><td>PCR cleanup kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>pluronic F-127</td><td>Anatrace</td><td>P305</td><td></td></tr><tr><td>polyethylene tubing PE-20</td><td>BD Intramedic</td><td>427406</td><td></td></tr><tr><td>polyethylene tubing PE-60</td><td>BD Intramedic</td><td>427416</td><td></td></tr><tr><td>Q5 Mastermix</td><td>New England Biolabs</td><td>M0492S</td><td></td></tr><tr><td>rare earth magnet 0.5&quot; OD 0.25&quot; ID</td><td>National Imports</td><td>NSN0814</td><td></td></tr><tr><td>rare earth magnet 0.75&quot; OD 0.5&quot; ID</td><td>National Imports</td><td>NSN0615</td><td></td></tr><tr><td>reverse primer: biotin-TGACCATTAGATACATTTCGC</td><td>Integrated DNA Technologies</td><td>n/a</td><td></td></tr><tr><td>syringe pump</td><td>Kent Scientific</td><td>Genie Plus</td><td></td></tr><tr><td>&amp;beta;-Casein from bovine Milk</td><td>Sigma-Aldrich</td><td>C6905</td><td></td></tr></tbody></table>

parallel high throughput single molecule kinetic assay for site-specific dna cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

Using this technique, the SSDC rates of NdeI were measured for a range of protein concentrations (0.25&#8211;4.00 U/mL) at two different concentrations of magnesium (2 mM and 4 mM). Each of these conditions was replicated at least twice, with a few hundred to 1,000 tethered DNAs per experiment. Figure 2 describes the experimental design. Figure 3 shows examples of data collection and analysis details. Figure 4 illustrates how the ra...

Watch this Scientific Journal Video about Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage at JoVE.com

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

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3.9K Views.  Emmanuel College. A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Video: Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Chemistry

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Genetics

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, ...

Biochemistry

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

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 ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Quantifying DNA Double Strand Break Repair with Nanoluciferase Reporter Assays

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli.
Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence.
These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Author Spotlight: Developing Novel Anticancer Therapeutics Targeting the DNA Damage Response

We present protocols for the assessment of double strand break repair pathway proficiency in cells using a suite of luminescence-based extrachromosomal reporter substrates.

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is ...

Cancer Research

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 ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

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Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

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

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

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

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

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