We thank Tony Kulesa, Robin Kirkpatrick and Evangelos Gatzogiannis for assistance with initial instrumentation setup and testing. We further thank Tony Kulesa for significant help writing and evaluating the customized single particle tracking software. This work was supported by startup funding and a Career Award at the Scientific Interface (to PCB) from the Burroughs Wellcome Foundation.

1. Preparation of biotin- &#955;-DNA
NOTE: Biotin-&#955;-DNA molecules are prepared by ligating a biotin-labeled oligonucleotide, 5&#39;-AGGTCGCCGCCC(A)20-biotin-3&#39; to&#160;&#955;-DNA molecules20.
<ol>
	<li>Prepare 0.1 mM biotin labeled oligo in TE buffer.</li>
	<li>Heat 0.5 mg/mL &#955;-DNA stock at 65 &#176;C for 60 s, and plunge into wet ice right away. 
	NOTE: Fast quench cooling reduces &#955;-DNA concatemerization.</li>
	<li>Pipette 100 &#181;L of the &#955;-DNA solution into a microcentrifuge tube.</li>
	<li>Add 1 &#181;L of the 0.1 mM oligo into 9 &#181;L of TE buffer.</li>
	<li>Add 2 &#181;L of the oligo solution to the microcentrifuge tube containing the &#955;-DNA. Mix thoroughly. 
	NOTE: Oligo is present in approximately a 12-fold molar excess over the complementary &#955;-DNA end.</li>
	<li>Heat the &#955;-DNA/oligo mixture at 65 &#176;C for 60 s.</li>
	<li>Slowly cool the mixture to room temperature. 
	NOTE: This step allows the oligo to anneal to the complementary &#955;-DNA end.</li>
	<li>Place the mixture on ice. Add 11 &#181;L of T4 DNA ligase reaction buffer (final buffer concentration 1x). Mix gently. Then add 2 &#181;L (800 units) of T4 DNA ligase enzyme. Mix gently.</li>
	<li>Incubate the mixture for 2 h at 16 &#176;C or overnight at 4 &#176;C.</li>
	<li>Purify the product using centrifugal filter tubes with a nominal molecular weight limit of 100 kDa. Specifically, transfer the step 1.9 mixture into a centrifugal filter tube and centrifuge at 14,000 x g for 5 min, wash with 400 &#181;L TE buffer twice, and collect the purified product. 
	NOTE: The as-purified biotin-&#955;-DNAs in TE buffer are stable at -20 &#176;C for up to one year.</li>
</ol>
2. Coverslip functionalization
NOTE: Glass coverslips are functionalized by reacting with silane-PEG-biotin. This one-step reaction protocol is simpler and more reliable in our experience than the standard two-step reaction protocol20. Coverslip functionalization with silane-PEG-biotin creates binding sites for streptavidin and minimizes nonspecific DNA and protein adsorption to the coverslip surface.
<ol>
	<li>Sonicate five no. 1 coverslips in 95% ethanol inside a staining jar for 10 min and then rinse with ultrapure water for three times. 
	NOTE: The fifth coverslip provides a spare in case of breakage.</li>
	<li>Fill the staining jar with 1 M KOH, sonicate for 10 min and then rinse with ultrapure water three times.</li>
	<li>Repeat the 2.1-2.2 cycle twice.</li>
	<li>Dry the coverslips under clean dry N2 gas flow.</li>
	<li>Further clean and dry the coverslips by applying air plasma treatment at 900 mTorr pressure for 5 min.</li>
	<li>Incubate coverslips with 25 mg/mL silane-peg-biotin dissolved in 95% ethanol at room temperature for 2 h. Specifically, sandwich 50 &#181;L silane-peg-biotin solution between two clean dry coverslips, and incubate the coverslip &#34;sandwiches&#34; inside a closed pipette tip box with about 10 mL of water at the bottom (not in contact with the coverslips) to minimize solvent evaporation.</li>
	<li>Wash away excess PEG molecules with ultrapure water and dry the PEG coated coverslips under clean dry N2 gas flow. 
	NOTE: The functionalized coverslips can be stored up to three days under ambient conditions.</li>
</ol>
3. Flow cell construction
NOTE: Flow cells are constructed by sandwiching a double-adhesive tape with pre-cut channels between a PEG functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated per flow cell.
<ol>
	<li>Design flow channels in a CAD software and cut the channels (the width, depth and length of each channels are 1, 0.13 and 12 mm, respectively) on a double-adhesive tape by using a tape cutter (typically eight channels are integrated per flow cell). Remove tape residuals to form the flow channels.</li>
	<li>To make PDMS slabs, thoroughly mix 45 g of PDMS with 5 g of crosslinking reagent (enough for making twelve 5-mm thick PMDS slabs which can be stored indefinitely under ambient conditions) using a mixer, pour the mixture into two 10 cm Petri dishes, and leave the dishes inside a vacuum chamber until essentially all air bubbles are gone (manually remove air bubbles if any remain when the dishes are removed from the vacuum chamber). Then transfer the dishes into an 80 &#176;C oven until PDMS solidifies (about 2 h).</li>
	<li>Cut out a PDMS slab that matches the size of the double-adhesive tape with pre-cut channels. Peel one protection film off the double-adhesive tape and adhere the film to a flat face of the PDMS slab. Punch the outlet and inlet holes on the PDMS slab using a biopsy hole-puncher with a 23 Gauge needle.</li>
	<li>Peel the second protection film off the double-adhesive tape and adhere the PDMS-tape assembly to the functionalized surface of the coverslip (make sure that the coverslip is flat. See a picture of a fully assembled flow cell in Figure 1a).</li>
</ol>
4. TIRF Imaging
NOTE: Biotin-&#955;-DNAs are tethered to a flow channel surface and laminar flow is applied to flow stretch the DNA molecules (Figure 1b). The &#62; 80% stretched DNA molecules serve as spatially extended templates for observing the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse TIRF imaging (Figure 1c &#38; e).
<ol>
	<li>Fix the flow cell on a microscope stage and load pre-degassed blank buffer (10 mM sodium phosphate, 2 mM NaCl, 50 &#181;M EDTA, 20 mM ethanol, 5% (v/v) glycerol, 0.01% Tween-20, 1% (v/v) &#946;-mercaptoethanol, pH 7.4), 0.2 mg/mL streptavidin solution, 1 mg/mL Bovine Serum Albumin (BSA) solution and 100 pM biotin-&#955;-DNA solution into four reservoirs that each have a Tygon tubing connected to a solenoid value and another Tygon tubing connected to a PEEK tubing by sleeving the Tygon tubing over the smaller PEEK tubing (prevents backflow of reagents). Degas small volumes of buffer by overnight exposure to vacuum or shorter exposure to vacuum with stirring (until bubbles are no longer visible).</li>
	<li>Insert the PEEK tubing of the blank buffer reservoir into one inlet hole of the flow cell. Prime the channel by flowing in blank buffer, and insert three other PEEK tubing into inlet holes The air pressure driven flow of each reagent is controlled by solenoid valves and fully automated via a computer script. Be careful not to induce air bubble formation in the channel.</li>
	<li>Connect a Tygon tubing with a short PEEK tubing from the outlet hole to a waste container. The short PEEK tubing at the outlet helps suppress air bubble formation during infusion by maintaining positive pressure inside the channel.</li>
	<li>Tethering biotin- &#955;-DNA molecules to a flow channel surface.
	<ol>
		<li>Flow in 0.2 mg/mL streptavidin solution, incubate for 5 min and then flow in blank buffer to wash out the unbound streptavidin.</li>
		<li>Flow in 1 mg/mL BSA solution, incubate for 1 min, and then flow in blank buffer to wash out the unbound BSA. 
		NOTE: BSA further suppresses DNA and sample adsorption to the surface.</li>
		<li>Flow in 100 pM biotin-&#955;-DNA solution, incubate for 10 min and then flow in blank buffer to wash out the unbound DNAs. 
		NOTE: After the DNA is bound to the surface, avoid rapid flows to prevent damage to the tethered DNA molecules.</li>
	</ol>
	</li>
	<li>To check the quality of the functionalized coverslip, flow in DNA staining dye such as Sytox orange under the assay conditions to be used (read 4.6 below), and start fluorescence imaging under 532 nm laser illumination. 
	NOTE: The quality of the functionalized coverslip is typically good and the density of flow stretched DNA molecules will be high. It is essential to have a high enough density of flow stretched DNA molecules so that DNA binding and sliding/1D diffusion events can be observed.</li>
	<li>Fine tune the TIRF angle to achieve best signal to noise ratio of images of stretched DNA molecules (Figure 1d).</li>
	<li>To record trajectories of single molecules moving along DNA, repeat steps 4.2-4.4 inside a new channel (without DNA staining dye), then infuse pre-degassed sub-nanomolar fluorophore labeled molecules at a high enough flow rate by using a syringe pump and collect time-lapse fluorescence images with a frame rate of 100 Hz. 
	NOTE: A flow rate equivalent to a Weissenberg number (Wi: the dimensionless product of the longest polymer relaxation time - approximately 0.2 s for &#955;-DNA - and the shear rate - the change in flow velocity with distance from the coverslip surface) of 500 inside the channel is recommended for single molecule imaging with a frame rate of 100 Hz21.
	<ol>
		<li>Collect 10,000 frames in one Field-Of-View (FOV) to produce a movie, and collect similar movies from multiple (typically ten) FOVs per sample. 
		NOTE: The density of single particles in one FOV should be neither too high - which will complicate data analysis by making object assignment across frames ambiguous, nor too low - which might lead to low occurrence of single molecule events on DNA.</li>
		<li>Optimize the illumination intensity so that molecules bound to the coverslip surface are quickly photo-bleached to suppress background while molecules diffusing on DNA are not bleached too quickly so that longer trajectories can be detected. 
		NOTE: We typically use 200 - 800 W/cm2 power density in the FOV.</li>
	</ol>
	</li>
	<li>At the end of step 4.6, infuse DNA staining dye to confirm that DNA molecules can still be flow-stretched.</li>
	<li>Run both positive and negative control samples. 
	NOTE: A good positive control is a tetrapeptide, TetraMethylRhodamine(TMR)-KRRR (TMR is coupled to the N-terminal amine) which has a mean 1D diffusion constant of 10.5 &#177; 0.7 (standard error) M(bp2/s) at neutral pH11. A good negative control is to measure labeled samples of interest in assay buffer in a channel that has no DNA tethered, where no on-DNA diffusion activity should be detected.</li>
</ol>
5. Data Analysis:
NOTE:&#160;A custom single particle tracking software is used to identify trajectories of single molecules diffusing along DNA and estimate diffusion constants. This software first determines the centroid positions of single particles with high accuracy, identifies particles that are stuck on the coverslip surface, and then links particles in different frames to form time-lapse trajectories. From these trajectories, the 1D diffusion constants are estimated.
<ol>
	<li>To start data analysis, open a scripting software (see table of materials) and go to the directory of the single particle tracking software. Open the script named &#34;largedataprocess3.m&#34;. One important parameter to be defined is the threshold value for single particle detection.</li>
	<li>To determine the optimal threshold value, run the &#34;Determine_threshold_value.m&#34; script. This script will indicate how the threshold value affects single particle detection.After determining the optimal threshold value, run the &#34;largedataprocess3.m&#34; script.</li>
	<li>After completion of single particle tracking analysis, filter the raw trajectories by running the &#34;Trajectory_filtering.m&#34; script. A Graphic User Interface (GUI) is built in to visualize the filtering step.</li>
	<li>On the GUI panel, set the minimal displacement along DNA, MinXDisp, to 2 pixels; set the maximal displacement transverse to DNA, MaxYDisp, to 2 pixels; set the minimal number of frames, minFrames, to 10; set the minimal number of states of triplet, minTriplets, to 10; set the minimal diffusion constant along DNA, D_par, to 0; set the maximal estimated diffusion constant transverse to DNA, D_trans, to 10 M(bp2)S-1; set the minimal statistic parameter, Chi2_stat, to -5; set the minimal ratio of displacement along DNA to that transverse to DNA, MinX/Y, to 2. 
	NOTE: All raw trajectories that pass these filtering parameters are listed in Table 1, and those that do not are listed in Table 3.</li>
	<li>Click on a trajectory number in Table 1, the trajectory will be displaced in Graphics 1 &#38;2. Click on the &#34;Play&#34; button to play the raw fluorescence images. Click on the &#34;Add_to_Table2&#34; to identify the trajectory as a single molecule sliding trajectory. In the end, all trajectories added to Table2 will be saved when closing the GUI.</li>
	<li>To calculate the mean diffusion constants, run the script &#34;Calculate_mean_diffusion_constant.m&#34;. The mean diffusion constant is estimated from the set of trajectories that have passed all filtering steps and been added to Table 2.</li>
</ol>

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The Broad Institute may elect to file patent applications that include aspects of the work presented here.

When switching from the traditional two-step coverslip functionalization reaction protocol to a one-step reaction protocol, we noticed that the reliability of coverslip functionalization is significantly improved. As the total hands-on time for coverslip preparation is less than thirty min, we recommend preparing fresh coverslips each day single molecule imaging is planned. To minimize deterioration of silane-PEG-biotin, the reagent should be aliquoted and stored under dry N2 gas at -20 &#176;C upon receipt. Here we do not use PEG molecules that produce -COO- or -CH3 terminal groups that have been used in the past20. The negative charges of -COO- groups and the hydrophobic -CH3 groups were utilized to prevent nonspecific adsorption of DNA molecules and specific protein samples to the surface, respectively. We observe very low surface adsorption by small peptide samples on the fully biotin-terminated PEG surface, while silane-PEG-COOH and/or silane-PEG-CH3 might be useful for assays of certain protein samples or when assay conditions (such as pH &#60;7.5) promote DNA-surface interactions.
The shift from glass slide flow cells to PDMS flow cells speeds up the flow cell&#160;construction and allows easy integration of multiple channels. We often engineer eight channels per flow cell to enable eight independent experiments per functionalized coverslip. To further increase throughput per coverslip, even larger numbers of micro-fabricated channels could be used.
It is advisable to prevent air bubble formation inside the channel at any point after initial filling. Usually, air bubbles do not cause loss of tethered DNAs (although this is possible), but rather disrupt flow and possibly cause DNAs to stick to the coverslip. Buffer degassing and the addition of small surfactant quantities are typically effective in preventing bubble formation within the tubing and flow channel. In cases where air bubbles are introduced to the tubing, try to flush the bubbles out under a slow flow and make sure that they do not flow through the region where DNAs are tethered.
A streamlined and efficient data analysis software is critical, due to the huge amount of data generated. We usually collect 700,000 high-resolution images from measurements on one flow cell, which can be processed using our custom single particle tracking software within one day on a desktop computer with a quad-core processor and 32 Gb memory. The false positive rate of detecting 1D diffusion trajectories by the software (step 5.5) is sample dependent and can be quite low given that: 1) The density of single particles in the FOV is low enough so that there is little ambiguity linking particles across frames (protein samples in general are more prone to nonspecific adsorption to the coverslip than peptide samples, and thus, the buffer and the power density for each protein sample needs to be optimized); 2) The signal to noise ratio of single particles is high enough so that the likelihood of false-identification of particles is low. Still, the software can make occasional mistakes, and we recommend manual inspection of the data using the provided GUI to evaluate the software performance. In an application particularly sensitive to false-positive trajectory detection, process parameters including the threshold value, minFrames, minTriplets, and Chi2_stat can be set more stringently at the cost of lower sensitivity for true binding events and potentially greater statistical bias in trajectory identification. We here share and describe our single particle tracking software with the hope of helping standardize data analysis methods and stimulate discussion about best practices.
In our protocol, which requires continuous flow during imaging to maintain the stretched state of the DNA template molecules, the fluid flow could bias the transport of some proteins along DNA templates if they diffuse by a hopping mechanism or if the hydrodynamic radius of the labeled fluorophore is large22,23. While such bias can be measured and corrected in most datasets, the transport of proteins labeled with a small fluorophore and diffusing by a sliding mechanism is not typically biased to a detectable extent by flow2,4,24. Notably, the impact of the flow velocity on the transport of proteins along DNA has been used to study the mechanisms of protein diffusing along DNA23.&#160;A closely related alternative approach that enables measurements in the absence of flow utilizes DNA templates with biotin at both termini that are transiently stretched in flow and tethered by both ends to the coverslip such that the DNA molecules remain in an extended configuration when the flow is turned off25,26,27,28.&#160;The double-tether approach has the advantage of allowing flow-free transport experiments but requires modifying both ends of the DNA template molecules, careful flow control during tethering, and characterizing the distances between the two tethered sites. In addition, the impact of heterogeneous tensions and DNA conformational dynamics across DNA molecules on the single-molecule assay must be assessed.
Altogether, in addition to studying 1D diffusion of molecules along DNA, the as-reported single molecule assay can benefit many in vitro biochemical and biophysical studies at the single molecule level including DNA target search processes by proteins, enzymatic activities on DNA, and more.
Troubleshooting
Problem 1: The flow channels leak.
Solution: First, make sure that the design of flow channels allows at least 1.5 mm margin for sealing around and between channels; and then, during assembly of the flow cell, make sure that contact surfaces between the coverslip, the double-adhesive tape and the PDMS slab are flat, dry and free of debris, that the pressure applied to form the seal is uniform, and that the sealing operation is performed at room temperature.
Problem 2: The density of tethered DNAs is too low.
Solution: This problem arises when the PEG functionalization efficiency is low or the DNA molecules are not functionalized with biotin. From our experience, the density of tethered DNA should be high for &#62; 90% of the coverslips prepared from fresh or properly stored silane-PEG-biotin reagent. In cases when the density of tethered DNA is low with a proven batch of biotin-&#955;-DNA, try boosting concentrations of silane-PEG-biotin during PEG functionalization and concentrations of streptavidin and biotin-&#955;-DNA during DNA tethering. If this fails, replace the PEG and streptavidin reagents.
Problem 3: DNAs stick to the coverslip and cannot be stretched by flow.
Solution: This problem can also arise&#160;when the PEG functionalization efficiency is low, and is aggravated by low assay pH. Try flowing in more BSA or raising the pH (eg to 8.0) to help suppress DNA adsorption to the surface (step 4.3.2). If DNA adsorption is a persistent issue under a particular assay condition, try including up to 90% silane-PEG-COOH during PEG functionalization.
DATA ANALYSIS NOTES:
We use custom single particle tracking software to identify trajectories of single molecules diffusing along DNA, from which their 1D diffusion constants, D1 are estimated. To improve data analysis, we implemented the radial symmetry algorithm18&#160;for localizing particles and the covariance-based estimator19&#160;for estimating 1D diffusion constants, which dramatically sped up data analysis without compromising accuracy. We previously validated the custom software by analyzing simulated data11. The major steps are described below.
Particle identification:
This step is for detecting and segmenting particles - diffraction-limited spots - from background. First, spatially band-pass filter the images to enhance the signal of particles in the 3-5 pixel range (which corresponds to the size of the point-spread function on our system) and then threshold based on intensity (see script: spt2_SD_sandbox.m).
Centroid assignment
Segmenting the image only localizes molecules to pixel-level spatial resolution. To localize molecules to much higher precision, employ the Radial Symmetry super resolution algorithm18&#160;to images filtered as above. (This method is based on an analytical calculation and thus vastly reduces computational burden compared with other popular high-accuracy methods such as 2D Gaussian fitting (see script: spt2_SD_sandbox.m).)
Particle tracking: 
To track the trajectory of a particle through time, we must re-identify the same particles as they move between frames. We consider the initiation, blinking, and termination stages to be components of a trajectory. We constrain linkage of particles in current frames to those appearing in previous frames by the maximum displacement allowed per frame. In cases where multiple linkages are possible, the Jonker-Volgenant algorithm is used to yield globally optimal linkage sets (see script: traceTraj4_kx.3.m).
D1 estimation: 
To estimate D1 values from trajectories, we use the Covariance-Based Estimator (CVE)19.&#160;Based on an analytical calculation, this method is more computationally efficient and statistically rigorous than other commonly used methods such as regression of mean squared displacements (see script: runspt5_SD.m).
Trajectory Filtering:
Some trajectories contain artifacts such as linkage to surface-bound molecules and must be removed. We implemented several features such as minimum displacement parallel to DNA, maximum displacement transverse to DNA, minimal length of trajectory, minimal number of triplet consecutive detected positions, and minimum probability score (see Xiong et al11 for definition) that can be filtered to isolate a minimally biased set of trajectories likely to represent molecules diffusing on DNA (see script: sptViewFig2_update3x.m).

A long-standing problem in biology is how endogenous proteins that act at specific sites in the genome can locate their DNA targets quickly enough for the organism to survive and respond effectively to its environment. Studies over the past forty years proposed and largely support the hypothesis that the kinetics of DNA target search by a protein can be accelerated by facilitated diffusion in which the protein alternates between 3D diffusion in the bulk and 1D diffusion (including sliding and hopping processes) along the DNA1. It is now known that many proteins involved in gene regulation, nucleic acid metabolism, and other processes are capable of sliding on DNA2,3,4,5,6,7,8,9. Furthermore, recent studies reported that even small peptides can bind to and slide on DNA, with the ability to carry a cargo; for example, a protein molecule or PCR primer, along DNA10,11,12,13,14.
Over the last 15 years, the single molecule flow-stretching assay has been widely used to study binding and diffusion of molecules along DNA2,15,16.&#160;In this type of assay, biotinylated double-stranded DNA molecules are immobilized to the surface and a laminar flow is applied to flow stretch the DNA. The &#62; 80% stretched DNAs serve as spatially extended templates for studying binding and transport activity of molecules labeled with fluorophores where the trajectories of single molecules along DNA are tracked by time-lapse fluorescence imaging. In our implementation, optimized for reproducibility and ease of use, this assay consists of five major steps: preparation of biotin-&#955;-DNA, coverslip functionalization, flow cell construction, fluorescence imaging and data analysis. In previous protocols17, glass coverslips were functionalized&#160;by first reacting with (3-Aminopropyl)triethoxysilane (APTES) and then with amine-reactive polyethylene glycol (PEG) reagents (e.g., NHS-PEG-biotin) to form a PEG layer that resists nonspecific adsorption of assay components to the coverslip surface. The quality of functionalized coverslips largely depended on the quality of PEG reagents and reaction conditions at each step. Our protocol describes a simplified functionalization protocol and multiplex flow cell construction which requires no liquid adhesive&#160;or curing time on the day flow cells are assembled. We also describe a streamlined and robust data analysis procedure11 that eliminates computationally intensive regression steps by applying a radial symmetry method for centroid localization18&#160;and a covariance-based diffusion constant estimator19.
Here, we report a simple, robust and high throughput single molecule flow stretching assay implementation with significant improvements made in coverslip functionalization, flow cell construction and data analysis. In particular, we developed a one-step coverslip functionalization protocol, in which clean dry coverslips react directly with silane-PEG-biotin. This protocol simplifies the coverslip preparation and improves the reliability of the quality of functionalized surfaces compared to the standard two-step reaction protocol. We describe the use of so-functionalized coverslips with multi-channel PDMS flow cells that enable robust tubing connections to be made without glue. These flow cells further include multiple computer-controlled inlets for each flow chamber which enable automated reagent flow to reduce hands-on time during setup and increased assay throughput.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="46">
			<td height="46" style="height:46px;width:249px;">Biotin labeled oligo, 5&#8217;-AGGTCGCCGCCC(A)20-biotin-3&#8217;</td>
			<td style="width:249px;">IDT</td>
			<td style="width:159px;">Custom oligo synthesis</td>
			<td style="width:249px;">Standard desalting purification is sufficient</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">&#955;-DNA</td>
			<td style="width:249px;">New England Biolabs Inc.</td>
			<td style="width:159px;">N3011S</td>
			<td style="width:249px;">Make aliquots and minimize freeze-thaw cycle number</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">T4 DNA ligase and ligation reaction buffer</td>
			<td style="width:249px;">New England Biolabs Inc.</td>
			<td style="width:159px;">M0202S</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Amicon centrifugal filter tubes with NMWL of 100k Dalton</td>
			<td style="width:249px;">EMD Millipore</td>
			<td style="width:159px;">UFC510008</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">No. 1 glass coverslips</td>
			<td style="width:249px;">VWR</td>
			<td style="width:159px;">48393-106</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Pure ethanol</td>
			<td style="width:249px;">VWR</td>
			<td style="width:159px;">89125-186</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Potassium hydroxide pellets</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:159px;">P250-500</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Plasma cleaner, model No. PDC-32G</td>
			<td style="width:249px;">Harrick Plasma</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:249px;">Silane-peg-biotin</td>
			<td style="width:249px;">Nanocs</td>
			<td style="width:159px;">PG2-BNSL-5k</td>
			<td style="width:249px;">Store at -20oC in dry nitrogen gas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">A CAD software</td>
			<td style="width:249px;">Autodesk</td>
			<td style="width:159px;">AutoCAD</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Double-adhesive tape</td>
			<td style="width:249px;">Adhesive Applications</td>
			<td style="width:159px;">8512-2-DP/DL</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Tape cutter, model No. CE5000-40-CRP</td>
			<td style="width:249px;">Graphtec America</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">PDMS and crosslink reagent kit</td>
			<td style="width:249px;">R.S. Hughes</td>
			<td style="width:159px;">RTV615 CLEAR 010</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Degassing chamber, model No. F42010-0000</td>
			<td style="width:249px;">BEL-ART Products</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Mixer, model No. ARV-310</td>
			<td style="width:249px;">Thinky</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:249px;">Hole puncher, Harris Uni-Cores, Size 0.5 mm</td>
			<td style="width:249px;">Harris Uni-Cores</td>
			<td style="width:159px;"></td>
			<td style="width:249px;">This can be substituted with a manual biopsy punch with a similar needle size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Tygon tubing</td>
			<td style="width:249px;">Cole Parmer</td>
			<td style="width:159px;">EW-06420-02</td>
			<td style="width:249px;">0.020&#34; ID, 0.060&#34;OD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Peek tubing</td>
			<td style="width:249px;">Western Analytical</td>
			<td style="width:159px;">PK007-031-Y</td>
			<td style="width:249px;">0.007&#34; ID, 0.031&#34;OD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Streptavidin</td>
			<td style="width:249px;">New England Biolabs Inc.</td>
			<td style="width:159px;">N7021S</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Bovine serum albumin (BSA)</td>
			<td style="width:249px;">New England Biolabs Inc.</td>
			<td style="width:159px;">B9000S</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Sytox orange nucleic acid stain</td>
			<td style="width:249px;">Invitrogen</td>
			<td style="width:159px;">S11368</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Ti-E Microscope</td>
			<td style="width:249px;">Nikon</td>
			<td style="width:159px;"></td>
			<td rowspan="5" style="width:249px;">Microscope, camera, objective, dichroic mirror and bandpass filter can be substituted for similar equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">100x Objective, NA/1.49, CFI Apo TIRF</td>
			<td style="width:249px;">Nikon</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">CMOS camera</td>
			<td style="width:249px;">Hammamatsu</td>
			<td style="width:159px;">Orca Flash 4.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Dichroic mirror</td>
			<td style="width:249px;">Semrock</td>
			<td style="width:159px;">FF560/659-Di01-25x36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Bandpass filter</td>
			<td style="width:249px;">Semrock</td>
			<td style="width:159px;">FF560/659-Di01-25x36</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">532 nm laser</td>
			<td style="width:249px;">Rayscience Innovation Limited, Shanghai</td>
			<td style="width:159px;">OEMLL-FN-532nm-C</td>
			<td style="width:249px;">Select wavelength to correspond to stain of choice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Syringe pump</td>
			<td style="width:249px;">Chemxy</td>
			<td style="width:159px;">Fusion 200</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">TMR-KRRR, &#62;=85% purity</td>
			<td style="width:249px;">MIT Biopolymer lab</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">pVIc-Cy3B, HPLC purified</td>
			<td style="width:249px;">A gift from Dr. Walter Mangel at Brookhaven National lab</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Script programming software</td>
			<td style="width:249px;">MathWorks</td>
			<td style="width:159px;">Matlab</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:249px;">* see parts list for solenoid valves, computer interface, and assembly instructions at: https://sites.google.com/site/rafaelsmicrofluidicspage/valve-controllers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 2a shows the detected trajectories of pVIc-Cy3B (pVIc: GVQSLKRRRCF; Cy3B is conjugated to the cysteine residue) molecules diffusing on DNA in 2 mM NaCl buffer at pH 6.5. The diffusion of pVIc is bi-directional and driven by ambient thermal energy. From these trajectories, the 1D diffusion constants (D1 values) are estimated for each trajectory (see the histogram in Figure 2b). The mean D1 value of this pVIc conjugate is calculated to be 22.2 &#177; 0.9 (standard error) M(bp2/s) by averaging each D1 value across trajectories weighted by the number of triplet consecutive position calls contained in each trajectory.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55923/55923fig1.jpg" /> 
Figure 1: Apparatus for tracking motion of single molecules along DNA. 
a) An eight-channel PDMS microfluidic chip adhered to a functionalized coverslip to which DNA can be tethered (with USA quarter coin for scale); b) Flow cell zoom schematic showing flow-stretched &#955;-DNA molecules; c) Schematic of TIRF microscope and flow system for stretching DNA; d) TIRF image of a flow-stretched &#955;-DNA; e) TIRF image of pVIc-Cy3B (pVIc: GVQSLKRRRCF; Cy3B is conjugated to the cysteine residue) molecules bound to a flow-stretched &#955;-DNA. This figure has been modified with permission2,12. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55923/55923fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55923/55923fig2.jpg" /> 
Figure 2: One-dimensional diffusion of pVIc (GVQSLKRRRCF) along DNA. 
a). Diffusion of pVIc along flow stretched &#955;-DNAs (137 trajectories) in 2 mM NaCl buffer at pH 6.5. x(t) and y(t) are the displacements along and transverse to &#955;-DNA, respectively. Dotted horizontal lines indicate missing points resulting from dye blinking. b). Histogram of the diffusion constant, D1 for pVIc diffusing along &#955;-DNAs. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55923/55923fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA at JoVE.com

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

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, ...

a-simple-robust-high-throughput-single-molecule-flow-stretching-assay

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.0K Views.  Massachusetts Institute of Technology and Harvard Medical School. This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

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

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

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

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

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 ...

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 ...