This work was supported by a grant from the National Institutes of Health (AI082190 to T.J.D.) and the California Institute for Regenerative Medicine (TB1-01186 and CL1-00502).

Manual Screening or IC50 Determination
This protocol can be used to determine the relative potency of a compound (IC50 value) by preparing a dilution series of the compound, or to manually screen for small-molecule inhibitors at a single concentration.
1. Preparation of Buffers, Reagents,&#160;and Required Instrumentation
<ol>
	<li>Prepare 50 ml&#160;of assay buffer (40 mM HEPES, pH 7.4 and 0.01% Tween-20) and filter sterilize. The buffer can be stored at room temperature (RT) for several months.</li>
	<li>Prepare a 70 nM working dilution of recombinant botulinum neurotoxin/A light chain (LC/A (1-425))17 in assay buffer, gently vortex, and store on ice. Testing of each compound requires at least 120 &#956;l of LC/A working dilution.</li>
	<li>Prepare 7 &#956;M FRET-based BoNT/A LC substrate working dilution in assay buffer containing 10% DMSO. Initially dilute a 5 mM stock solution of the substrate in an appropriate volume of 100% DMSO then slowly (drop-wise) add the assay buffer while gently mixing. Wrap in foil and store on ice. Testing of each compound requires at least 130 &#956;l of the substrate working dilution.</li>
	<li>Configure a fluorescence microtiter plate reader to shake the plate at medium speed for 10 sec, then monitor the fluorescence at excitation and emission wavelengths of 490 nm and 523 nm, respectively, every 5 min (kinetic mode) for 90 min at RT. Note that product formation (i.e.&#160;the increase in fluorescence) is linear throughout this period of time (Figure 1A).</li>
</ol>
2. Preparation of Compound Dilution Series
<ol>
	<li>Determine the compound concentration range (i.e.&#160;number of dilutions in the dilution series and dilution factor); the concentration range is chosen based on the expected IC50 value to ensure an optimal curve fit. For compounds with an unknown IC50, use undiluted compound stock as the highest compound concentration.</li>
	<li>Label tubes with the appropriate compound name and concentration.</li>
	<li>Aliquot 100% DMSO to all of the tubes to prepare them for compound serial dilution. A minimum of 2 &#956;l&#160;final dilution volume is recommended, where 1 &#956;l of each concentration is used per well in the assay plate. For example, a standard 1:3 dilution series requires mixing 2 &#956;l of compound with 4 &#956;l DMSO per tube.</li>
	<li>Prepare serial dilutions of compounds and vortex or manually mix thoroughly in between each dilution; change pipette tips in between each dilution. For the standard 1:3 dilution series, add 2 &#956;l of the undiluted compound to 4 &#956;l of DMSO in the first tube of the dilution series and mix well. With a new pipette tip, remove 2 &#956;l of the first dilution and add to 4 &#956;l of DMSO in the second tube; mix well. Repeat for the remaining dilutions. Make sure to mix the compound dilutions well to ensure that the compound concentrations are accurate.</li>
	<li>Cover the compound tubes and set aside at RT.</li>
</ol>
3. Plate Preparation and Spotting (Table 1)
<ol>
	<li>Obtain a flat-bottom, opaque, black 96-well plate. See the recommended plate setup in Table 1, where each compound dilution series is formatted in rows of a 96-well plate. Manually dispense 1 &#956;l of the respective compound dilution directly to the bottom of each corresponding well (wells 1-9). Dispense 1 &#956;l of the highest concentration of compound to be tested to well 11 (compound intrinsic fluorescence control).</li>
	<li>Manually dispense 1 &#956;l of a known potent inhibitor to well 12 (positive control), and 1 &#956;l 100% DMSO to well 10 (negative control).</li>
	<li>With an automated pipettor, dispense 79 &#956;l of assay buffer to the side of wells 1-10 and 12 (compound dilutions and positive control), and 89 &#956;l to the side of well 11 (compound intrinsic fluorescence control). Make sure not to touch the tip to the bottom of the well where compound is present&#160;so that you do not cross-contaminate the wells.&#160;</li>
	<li>Vortex the LC/A working dilution and use an automated pipettor to add 10 &#956;l to the side of each well, except for well 11 (compound intrinsic fluorescence control). Gently tap the plate to ensure that all of the added enzyme goes to the bottom of the well.</li>
	<li>Gently&#160;mix, cover,&#160;and incubate the plate for 5 min at RT. This incubation provides a pre-equilibrium step for the compounds to bind to the LC/A prior to substrate addition.</li>
</ol>
4. Substrate Addition and Fluorescence Measurement
<ol>
	<li>Gently vortex the BoNT/A LC substrate, and using an automated pipettor add 10 &#956;l of substrate to the other side of each well. Be sure to add the substrate to the side of the well opposite of where the enzyme was added earlier.</li>
	<li>Gently tap the plate to mix the reagents, immediately place in plate reader and begin fluorescence measurement with the settings previously specified in step 1.4.</li>
	<li>The enzyme velocities are calculated from the slope of the line generated by graphing the relative fluorescence unit (RFU) vs. time during the linear period of the reaction.</li>
</ol>
Table 1.&#160;Suggested assay plate layout.
<table border="1">
	<tbody>
		<tr>
			<th>Well #</th>
			<th>Well description</th>
			<th>Compound</th>
			<th>Assay Buffer</th>
			<th>Substrate</th>
			<th>LC</th>
			<th>DMSO</th>
		</tr>
		<tr>
			<td>1</td>
			<td>Compound dose 1</td>
			<td>1 &#956;l</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l&#160;</td>
			<td>---</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Compound dose 2</td>
			<td>1 &#956;l</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l</td>
			<td>---</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Compound dose 3</td>
			<td>1 &#956;l</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l</td>
			<td>---</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Compound dose 4</td>
			<td>1 &#956;l</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l</td>
			<td>---</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Compound dose 5</td>
			<td>1 &#956;l</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l</td>
			<td>---</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Compound dose 6</td>
			<td>1 &#956;l</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l</td>
			<td>---</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Compound dose 7</td>
			<td>1 &#956;l</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l</td>
			<td>---</td>
		</tr>
		<tr>
			<td>8</td>
			<td>Compound dose 8</td>
			<td>1 &#956;l</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l</td>
			<td>---</td>
		</tr>
		<tr>
			<td>9</td>
			<td>Compound dose 9</td>
			<td>1 &#956;l</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l</td>
			<td>---</td>
		</tr>
		<tr>
			<td>10</td>
			<td>Negative control 
			(100% LC activity)</td>
			<td>---</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l</td>
			<td>1 &#956;l</td>
		</tr>
		<tr>
			<td>11</td>
			<td>Compound intrinsic fluorescence control</td>
			<td>1 &#956;l</td>
			<td>89 &#956;l</td>
			<td>10 &#956;l</td>
			<td>---</td>
			<td>---</td>
		</tr>
		<tr>
			<td>12</td>
			<td>Positive control 
			(0% LC activity)</td>
			<td>1 &#956;l</td>
			<td>79 &#956;l</td>
			<td>10 &#956;l</td>
			<td>10 &#956;l</td>
			<td>---</td>
		</tr>
	</tbody>
</table>
Automated Operation for High-Throughput Screening
5. Preparation of Buffers, Reagents, Compound Plates, Barcodes, and Required Instrumentation
<ol>
	<li>Thaw the sealed stock compound plates at room temperature for at least 1 hr.</li>
	<li>Prepare 500 ml (or more, depending on the number of compounds to screen) of the assay buffer (40 mM HEPES, pH 7.4 and 0.01% Tween-20) and filter sterilize. The filtered assay buffer can be stored at RT for several months.</li>
	<li>Prepare an 8.75 nM working dilution of recombinant BoNT/A LC (1-425) in assay buffer, vortex, and store on ice. Each well in a low-volume 384-well plate requires 8 &#956;l. Prepare a 10-15% excess, or approximately 3.7 ml or each low-volume 384-well plate.</li>
	<li>Prepare a 3.5 &#956;M FRET-based BoNT/A LC substrate working dilution in assay buffer containing 10% DMSO. Initially dilute the 5 mM stock solution of the substrate directly in the appropriate volume of 100% DMSO, then slowly (drop-wise) add the assay buffer while gently mixing. Wrap in foil and store on ice. Each well in a low-volume 384-well plate requires 2 &#956;l. Prepare a 10-15% excess, or approximately 1 ml for each low-volume 384-well plate.</li>
	<li>Prepare barcodes for each black, 384-well, low-volume microtiter plate [assay plate] and adhere the label directly to the side of the plate where the barcode scanner can read it; make sure the label does not&#160;extend over the plate side, or it may interfere with plate handling and stacking. Stack the plates in numerical order and set aside. Cover the top plate. Note that this step is optional, but facilitates tracking results to corresponding compounds when multiple plates are used.</li>
	<li>Centrifuge the thawed, sealed stock compound plates at 200 x g for 30 sec at RT. Carefully remove the plate seal, make sure to remove any remaining adhesive, and cover the plate with a clean plastic lid to prevent evaporation. Note that if any adhesive is present on the plate surface, it may interfere with automated lid transfer and plate stacking. Place the covered plate in a hotel attached to the liquid handler. Do not keep the plates uncovered or evaporation will occur.</li>
	<li>Configure a stacked fluorescence microtiter plate reader to read the fluorescence of each assay plate at excitation and emission wavelengths of 490 nm and 523 nm, respectively, every 30 min (kinetic mode) for 3-6 cycles at RT.</li>
</ol>
6. Preparation of Pin Tool
<ol>
	<li>Visually inspect the 384-head pin tool for any abnormalities, including warping, splintering or large debris.</li>
	<li>Fill three separate trays with approximately 1 in of 100% methanol (HPLC grade), 100% isopropanol (HPLC grade), and pin tool cleaning solution diluted in ultrapure water.</li>
	<li>Fill the cleaning station on a liquid handler with 100% DMSO.</li>
	<li>Stack 2-3 pieces of clean, new blotting paper in a tray. It is important to calibrate the robot in the z-axis so that the pins come in contact with, but do not puncture, the blotting paper.</li>
	<li>Secure a fan attachment to the working area for drying the pins after washing.</li>
	<li>Attach the pin tool to robotic arm of the liquid handler and preclean the pins by dipping in DMSO, drying on blotting paper, soaking for 2 min&#160;in cleaning solution, drying on blotting paper, dipping in isopropanol, drying on blotting paper, dipping in methanol, drying on blotting paper, and finally drying over the fan.</li>
</ol>
7. Preparation of Low-volume Liquid Dispenser
<ol>
	<li>Attach a low-volume (1-10 &#956;l) cassette to the liquid dispenser, and connect a waste line to an appropriate vessel.</li>
	<li>Visually inspect the dispensing nozzles of the cassette and check that they are not warped, torn or blocked. It&#39;s important to keep the channels and nozzles clean as precipitation or debris will influence liquid distribution.</li>
	<li>Prime the channels by running 30 ml of ultrapure water, 30 ml of 70% ethanol, then 30 ml of ultrapure water again directly into waste vessel.</li>
	<li>Position a low-volume 384-well assay plate into the liquid dispenser and add a known volume of water to each well. Visually inspect to make sure each well is receiving the correct volume and there is no spilling or solution dispensed in between the wells.</li>
	<li>Prime the tubes with air for approximately 5 sec to remove the water and dry the channels.</li>
</ol>
8. Dispensing BoNT/A Light Chain into Assay Plate
<ol>
	<li>Submerge the liquid dispenser intake line into BoNT/A LC working solution, prime to remove any air, and dispense 8 &#956;l into each well of a low-volume 384-well assay plate. Note that the plates can be manually positioned on the working area of the liquid handler, or automatically positioned and restacked using an attached plate hotel.</li>
	<li>Cover each plate with a clean plastic lid and stack in a different column of the plate hotel attached to the liquid handler. Do not keep plates unsealed or evaporation will occur.</li>
	<li>Clean the liquid dispenser by running 30 ml&#160;of ultrapure water, 30 ml&#160;of 70% ethanol, 30 ml&#160;of ultrapure water again directly into waste vessel.</li>
	<li>Prime the tubes with air for approximately 5 sec to dry the channels.</li>
</ol>
9. Stamping Compounds into Assay Plate
<ol>
	<li>Assign areas on the liquid handler platform for the compound plates, assay plates and containers with cleaning solutions.</li>
	<li>Program the liquid handler to place the compound plates and the assay plates in the designated positions and remove the plate lids prior to stamping.</li>
	<li>Stamp 50 nl&#160;of the compound directly into the assay plate containing 8 &#956;l of BoNT/A LC. When calibrating the stamping program, make sure that the pins are submerged, but do not scratch the bottom of the assay plate. Note that the addition of compounds in 100% DMSO may create a local zone of enzyme denaturation as it disperses. Care should therefore be taken to assess the level of enzyme denaturation by comparing the DMSO only control to the untreated control (i.e.&#160;enzyme without the addition of DMSO).</li>
	<li>Clean the pins after each plate by dipping in DMSO, drying on blotting paper, dipping in isopropanol, drying on blotting paper, dipping in methanol, drying on blotting paper, and finally drying over the fan.</li>
	<li>After the compounds have been stamped in the assay plate with enzyme, separately stack the stock compound plates and the assay plates. Cover the top assay plate in the stack to prevent evaporation and set aside. Make sure to incubate LC with compounds for at least 5 min at RT; longer incubation times can be used when stamping a large number of plates.</li>
</ol>
10. Dispensing Substrate into the Assay Plate
<ol>
	<li>Submerge the intake line of the liquid handler into the BoNT/A LC substrate working solution and protect from light. Check the dispensing nozzles to ensure that they are dry and not blocked. Prime the channels to remove all air bubbles.</li>
	<li>Un-stack the assay plates and dispense 2 &#956;l of working dilution of the substrate into each well. Check that the substrate is being dispensed directly into each well. Stack the plates and place one empty assay plate on top of the first plate and one empty plate under the bottom plate in the stack to prevent evaporation during reading and restacking in plate reader.</li>
</ol>
11. Fluorescence Measurements
<ol>
	<li>Immediately load the stack of assay plates into a fluorescence plate reader.</li>
	<li>Begin the fluorescence measurement with the settings previously specified in step 5.7. The assay rate for each compound is calculated from a graph of &#160;the relative fluorescence unit (RFU) over time. Note that it is important to include a negative control (DMSO) to obtain an uninhibited rate; a positive control with a known inhibitor can also be added to each plate. A representative output from a single time point is shown in Figure 2.</li>
</ol>
12. Cleaning up
<ol>
	<li>Seal each compound stock plate with a foil seal using a roller, and store at -80 &#176;C.</li>
	<li>After each assay plate has been read, discard the plates, any remaining LC/A or substrate dilutions in the biohazard waste.</li>
</ol>

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The authors have nothing to disclose.

The FRET-based BoNT/A LC assay described here represents an attractive method for identifying and characterizing small molecules that modulate BoNT/A LC activity. The solution-based nature of the assay makes this protocol amenable to high-throughput screening described in the Automated Operation section of the Protocol Text. The Z-factor is often used to determine the suitability of an assay for HTS campaigns18. Determined as Z = 1 &#8211; 3 (&#963;p + &#963;n)/(&#956;p - &#956...

Botulinum neurotoxin A (BoNT/A), the most potent toxin currently known (LD50 ~1 ng/kg)1, is a potent neurotoxin that is produced by the bacterium Clostridium botulinum. Within an affected host, BoNT/A disrupts neurotransmission at the neuromuscular junction by binding motor neurons, internalizing into the cytosol, and ultimately cleaving neuronal proteins that are essential for acetylcholine exocytosis. Once inside a neuron, BoNT/A can persist for as long as several months2. Long-term inhibition of acetylcholine release hampers normal muscle contraction and results in flaccid paralysis, which, in severe cases, may result in cardiac and/or respiratory failure. Because of its extreme potency, ease of production, and long-term effects within the host, the CDC has labeled all BoNT serotypes as high-risk bioterrorism agents.
The mechanism of action of the toxin involves numerous steps, including binding to neuronal surface receptors, cellular uptake via receptor-mediated endocytosis, and translocation into the neuron cytosol. BoNT/A is comprised of two chains, a heavy and light chain, and both chains are required for toxicity. The heavy chain (HC) contains binding and translocation domains, while the light chain (LC) is a zinc-dependent metalloprotease that translocates from the endosome to the cytoplasm. Once inside the cytosol, the LC/A metalloprotease localizes to the inner cytoplasmic membrane and cleaves the membrane-bound host protein SNAP-25. SNAP-25 is a member of the SNARE (Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor) protein family, which plays a crucial role in acetylcholine exocytosis (reviewed in reference3). LC/A cleavage of SNAP-25 impairs SNARE complex function, which inhibits acetycholine neurotransmitter release and impairs muscle contraction.
Currently, treatment for botulism is limited and often includes administration of an equine neutralizing antibody4; however, because the toxin is rapidly internalized into neurons, the antibody has the antibody has a narrow time window of administration. Thus, many researchers believe that the BoNT/A LC may be a better therapeutic target. Because the LC is a zinc-dependent metalloprotease, one approach to inhibit LC/A activity has been to develop compounds that chelate the active-site zinc ion. For example, hydroxamate compounds chelate the active-site zinc and have excellent in vitro potency (Ki of the best BoNT/A LC small molecule inhibitor to date is 77 nM)5. However, many small molecules fail to advance as therapeutics due to various problems ex vivo or in vivo, including poor aqueous solubility, rapid metabolism, and/or high cytotoxicity. Therefore, new compounds with improved pharmacological and pharmacokinetic properties are needed. Small molecule compound identification often involves high-throughput screening (HTS) to identify novel scaffolds. Initial methods for BoNT/A LC activity screening were based on HPLC detection of short peptide substrate cleavage, which is time-consuming and not amenable to HTS applications6-8. Subsequently, Schmidt and colleagues9 developed a high-throughput BoNT/A LC activity assay that utilizes a fluorescein-labeled peptide substrate covalently attached to a microtiter plate. The BoNT/A LC cleaves the substrate and releases fluorescein, which can be quantified with a fluorometer. The plate format of this assay allows numerous compounds to be screened simultaneously; however, the assay requires labeling synthetic peptides with fluorescein and coating the assay plates with derivatized substrate molecules, which are cumbersome techniques. A much simpler method for detecting BoNT/A LC activity at low concentrations was later described by Schmidt et al., where a series of fluorogenic substrates were utilized to monitor BoNT LC activity in real time10. Additional techniques described in the literature include a depolarization after resonance energy transfer-based assay to detect and quantify BoNT activity in crude extracts; this method can be used for high-throughput applications10,11, although it requires sophisticated equipment to measure fluorescence resonance energy transfer (FRET) and polarization signals. Finally, several cell-based models for BoNT intoxication have been reported (reviewed in reference11) that will enable researchers to study the often limiting properties of compounds previously mentioned, including cytotoxicity, cell permeability, and stability. However, most of the existing cell-based assays are not amenable to HTS, and are labor and time intensive.
Herein, we describe a detailed protocol for a HTS method that utilizes the commercially available FRET-based BoNT/A LC substrate. The substrate is based on the SNAP-25 cleavage sequence and is a synthetic 13-mer peptide that contains a terminal fluorophore and quencher. BoNT/A cleavage separates the fluorophore and quencher, abolishing FRET and increasing measured fluorescence, which can be continually measured in a fluorometer plate reader. The assay is used routinely in our, as well as other laboratories, to identify new classes of BoNT/A LC inhibitors or to determine the relative potency of previously identified compounds5,12-15. This assay is suitable for HTS because of its simplicity, automation potential, low cost of materials, and ability to screen numerous compounds simultaneously (see reference16; Cagli&#269; et al., submitted; Bompiani&#160;et al., in preparation). In addition to HTS, this assay can be used to compare the relative potency of compounds by determining the IC50 value (concentration required to inhibit 50% of BoNT/A LC activity) of a compound. The assay can either be performed manually in a 96-well format (Manual Screening section of the Protocol Text) or can be automated in a 384-well format for HTS (Automated Operation section of the Protocol Text).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >HEPES</td>
			<td >Teknova</td>
			<td >H1021</td>
			<td ></td>
		</tr>
		<tr >
			<td >Tween-20</td>
			<td >Fisher Scientific</td>
			<td >BP337-100</td>
			<td ></td>
		</tr>
		<tr >
			<td >Methanol (HPLC-grade)</td>
			<td >Sigma-Aldrich</td>
			<td >34860</td>
			<td ></td>
		</tr>
		<tr >
			<td >Isopropanol (HPLC-grade)</td>
			<td >Sigma-Aldrich</td>
			<td >650447</td>
			<td ></td>
		</tr>
		<tr >
			<td >96-well Black assay plate</td>
			<td >Costar</td>
			<td >3915</td>
			<td ></td>
		</tr>
		<tr >
			<td >384-well Low-volume black assay plate</td>
			<td >Greiner</td>
			<td >788076</td>
			<td ></td>
		</tr>
		<tr >
			<td >SNAPtide FITC/Dabcyl substrate</td>
			<td >List Biological Laboratories</td>
			<td >521</td>
			<td >FRET-based BoNT/A LC substrate</td>
		</tr>
		<tr >
			<td >Pin cleaning solution</td>
			<td >V&#38;P Scientific</td>
			<td >VP 110</td>
			<td ></td>
		</tr>
		<tr >
			<td >Lint-free blotting paper</td>
			<td >V&#38;P Scientific</td>
			<td >VP 540DB</td>
			<td ></td>
		</tr>
		<tr >
			<td >Biomek Seal and Sample Aluminum foil lids</td>
			<td >Beckman Coulter</td>
			<td >538619</td>
			<td ></td>
		</tr>
	</tbody>
</table>

a high-throughput-compatible fret-based platform for identification and characterization of botulinum neurotoxin light chain modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.

The FRET-based BoNT/A LC assay can be performed manually in a low-throughput manner to characterize known inhibitors or screen small libraries; alternatively, the protocol can be scaled-down and automated for high-throughput screening (HTS) with large libraries with the aim of identifying novel BoNT/A LC inhibitors. Regardless of the approach taken, an increase in fluorescence should be observed over time when BoNT/A LC is incubated with the substrate (Figure 1A shows a representative plot of fluorescenc...

Watch this Scientific Journal Video about A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators at JoVE.com

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and ...

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5.3K Views.  The Scripps Research Institute. The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Video: A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

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

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

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

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

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Ethylene Polymerizations Using Parallel Pressure Reactors and a Kinetic Analysis of Chain Transfer Polymerization

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel &#945;-diimine ethylene polymerization catalyst. Initial polymerizations with the catalyst lead to optimized reaction conditions, including catalyst concentration, ethylene pressure and reaction time. Using gas-uptake data for these reactions, a procedure to calculate the initial rate of propagation (kp) is presented. Using the optimized conditions, the ability of the nickel &#945;-diimine polymerization catalyst to undergo chain transfer with diethylzinc (ZnEt2) during ethylene polymerization was investigated. A procedure to assess the ability of the catalyst to undergo chain transfer (from molecular weight and 13C NMR data), calculate the degree of chain transfer, and calculate chain transfer rates (ke) is presented.

A protocol for the high-throughput analysis of polymerization catalyst, chain transfer polymerizations, polyethylene characterization, and reaction kinetic analysis is presented.

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and flexibility of their emission properties can be achieved by controlling their composition and dopants. However, a lot of work is still required to improve their properties and to reduce the production cost. A possible approach is to correlate the luminescence properties of the Sialon particles with their local structural and chemical environment in order to optimize their growth parameters and find novel phosphors. For such a purpose, the low-voltage cathodoluminescence (CL) microscopy is a powerful technique. The use of electron as an excitation source allows detecting most of the luminescence centers, revealing their luminescence distribution spatially and in depth, directly comparing CL results with the other electron-based techniques, and investigating the stability of their luminescence properties under stress. Such advantages for phosphors characterization will be highlighted through examples of investigation on several Sialon phosphors by low-energy CL.

Low-energy Cathodoluminescence for OxyNitride Phosphors

An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL).

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...