Name: Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis (Video) | JoVE Journal
Uploaded: 2017-01-07T18:00:00.000+00:00
Duration: 8 min 9 s
Description: Learn MicroScale Thermophoresis for mapping DNA aptamer binding sites to small molecules like ATP. This method determines affinity and stoichiometry independent of partner size.

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5 minutes

The authors have no acknowledgements.

1. Preparation of the Aptamer Working Stock
<ol>
	<li>Follow the manufacturer&#39;s instructions and dissolve the oligonucleotide (5-Cy5-CCTG GGGGAGTATTGCGGAGGAAGG-3, sequence from reference18) in water, reaching a 100-&#181;M final concentration.</li>
	<li>Prepare the aptamer working solution by diluting the oligonucleotide stock to 200 nM with binding buffer (20 mM Tris, pH 7.6; 300 mM NaCl; 5 mM MgCl2; 0.01% Tween20).</li>
	<li>Incubate the mixture for 2 min at 90 &#176;C, let the sample immediately cool down on ice, and use the sample at room temperature.</li>
</ol>
2. Preparation of the Ligand Dilution Series
<ol>
	<li>For each ligand (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenine, guanosine triphosphate (GTP), cytosine triphosphate (CTP), deoxyadenosine triphosphate (dATP), and S-adenosyl methionine (SAM); 10 mM stock each), prepare a 16-step serial dilution in 200 &#181;l micro reaction tubes. 
	NOTE: Centrifugation of ligand stocks for 5 min at 14,000 x g may help to remove aggregates. Low volume, low binding reaction tubes are recommended to avoid adsorption of molecules to the tube walls.</li>
	<li>Start with a maximum concentration of at least 50 times higher than the estimated affinity and reduce the ligand concentration by 50% in each dilution step. 
	NOTE: The concentration finder tool implemented in the control software simulates binding data and helps with finding the right concentration range for the dilution series.</li>
	<li>Fill 20 &#181;l of the ligand stock (10 mM) in tube 1. Add 10 &#181;l of aptamer binding buffer into micro reaction tubes 2 to 16.</li>
	<li>Transfer 10 &#181;l of tube 1 to tube 2 and mix properly by pipetting up and down several times. Transfer 10 &#181;L to the next tube and repeat this dilution for the remaining tubes.</li>
	<li>Discard the 10 &#181;l excess from the last tube. Avoid any buffer dilution effects. The buffer in tube 1 and in tubes 2-16 must be identical.</li>
</ol>
3. Preparation of the Final Reaction Mix
<ol>
	<li>Prepare the individual binding reactions with a volume of 20 &#181;l (10 &#181;l of aptamer working solution + 10 &#181;l of the respective ligand dilution) to minimize pipetting errors. A volume of only 4 &#181;l is suf&#64257;cient to &#64257;ll the capillary.</li>
	<li>Add 10 &#181;l of the 200 nM aptamer working solution to 10 &#181;l of each ligand dilution and mix properly by pipetting up and down several times.</li>
	<li>Incubate the samples for 5 min at room temperature and &#64257;ll the samples into standard capillaries by dipping the capillaries into the sample. Longer incubation times may be necessary for some interactions; however, 5 min is adequate for most. Touch the capillaries only on the sides, NOT on the middle part, where the optical measurement will be taken.</li>
	<li>Place the capillaries onto the capillary tray and start the MST device.</li>
</ol>
4. Starting the MST Device
NOTE: The device provides two pre-installed software packages, the &#39;&#39;control&#34; software for the technical setup of the experimental conditions and the &#39;&#39;analysis&#34; software for the interpretation of the produced data.
<ol>
	<li>Before placing the capillary tray into the MST device, start the control software and adjust the overall desired temperature by selecting &#39;&#39;enable manual temperature control&#34; in the &#39;&#39;temperature control&#34; dropdown menu. Adjust the temperature to 25 &#176;C in this way. 
	NOTE: The MST instruments can be temperature-controlled from 22 to 45 &#176;C.</li>
	<li>Wait for the temperature to reach the expected level and then place the capillary tray into the MST device.</li>
	<li>Set the LED channel to &#39;&#39;red&#34; for Cy5 dyes and adjust the LED power to gain a &#64258;uorescence signal of 300 to 1,000 fluorescence units at the MST device with a standard sensor. 25% LED power is used in this study. 
	NOTE: 6,000 to 18,000 fluorescence units are recommended for the MST with a high-sensitivity sensor.</li>
</ol>
5. Capillary Scan
<ol>
	<li>Carry out a capillary scan to check different quality aspects of the sample by choosing the capillary position on the &#34;control&#34; software and clicking on &#34;start cap scan&#34; before starting the MST measurement.</li>
	<li>Inspect the capillary scan for fluorescence enhancement/quenching and sticking effects (U-shaped or flattened peaks) in the software. 
	NOTE: More details on the detection and handling of fluorescence and sticking effects can be found in the discussion.</li>
</ol>
6. MST Measurement
NOTE: Before starting the MST measurement, make sure to exclude sticking effects, enhancement/quenching effects, or pipetting errors, and ensure that the capillary scan indicates that the &#64258;uorescence signal is suf&#64257;cient. For more details, see the discussion.
<ol>
	<li>Assign the ligand concentrations from the dilution series to the respective capillary position in the &#39;&#39;control&#34; software. Consider the dilution step of mixing the aptamer and ligand (1:1).</li>
	<li>Enter the highest concentration of ligand (5 mM) for capillary #1, select the correct dilution type (here, 1:1), click on the maximum concentration, and use the drag function to automatically assign the remaining concentrations in capillaries #2-16. The lowest concentration is 152.6 nM.</li>
	<li>Enter the concentration of the &#64258;uorescent aptamer (here, 100 nM) in the respective section of the control software.</li>
	<li>Use the default settings, which detect the &#64258;uorescence for 5 sec, record the MST for 30 sec, and record the fluorescence for a further 5 sec after the inactivation of the laser to monitor the back diffusion of molecules.</li>
	<li>Adjust the laser power to 20% in the respective section of the control software. 
	NOTE: In order to receive the best signal-to-noise ratio and to avoid unspecific effects, a laser power of 20-40% is recommended. In specific cases, a higher laser power may be required to get a good separation of unbound and bound molecules.</li>
	<li>Save the experiment after selecting the destination folder and start the MST measurement by pressing the &#39;&#39;Start MST measurement&#34; button. 
	NOTE: The .ntp file will be generated in the destination folder. Using this setup, one measurement lasts 10-15 min.</li>
	<li>Repeat the experimental procedure at least twice for a more accurate determination of the EC50 value. 
	NOTE: In order to test the technical reproducibility, the same capillaries can be scanned several times (technical repeats).</li>
</ol>
7. MST Data Analysis
NOTE: The analysis software enables the analysis of data on the &#64258;y during the measurement. The analysis software plots the MST time traces and changes in the normalized &#64258;uorescence (Fnorm) versus the ligand concentration37.
<ol>
	<li>Start the MST analysis software (MO.Affinity Analysis) and load the .ntp file from the destination folder. Select &#34;MST&#34; as the analysis type in the data selection menu. 
	NOTE: In case of ligand-dependent fluorescence effects, the initial fluorescence can be chosen for analysis.</li>
	<li>Add the respective technical or biological run(s) to a new analysis by drag-and-drop or by pressing the &#34;+&#34; button below the respective experimental run.</li>
	<li>Press the information button below the respective experimental run to obtain information on the properties of the experiment, MST traces, capillary scan, capillary shape, initial fluorescence, and bleaching rate. 
	NOTE: These raw data can also be inspected in later steps of the analysis.</li>
	<li>Visually inspect the MST traces for aggregation and precipitation effects, visible as bumps and spikes. 
	NOTE: For more information on the detection and handling of aggregation effects, read the discussion.</li>
	<li>Visually inspect the capillary scan and the capillary shape overlay for adsorption effects, visible as flattened or U-shaped peaks. Visually inspect the capillary scan and the initial fluorescence for fluorescence effects. Visually inspect the bleaching rate for photobleaching effects.</li>
	<li>Switch to the dose-response mode and change the analysis setting to &#34;expert&#34; mode by pressing the respective button. Select &#34;T-Jump&#34; as the MST evaluation strategy.</li>
	<li>Select the &#34;Hill&#34; model for curve &#64257;tting. The binding parameters will automatically be calculated. Normalize the data by choosing the respective type of normalization in the &#34;compare results&#34; menu. Export the data either as an .xls or .pdf. 
	NOTE: The table below the binding graph summarizes the calculated binding parameters.</li>
</ol>

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<li>Jhaveri, S., Rajendran, M., Ellington, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+selection+of+signaling+aptamers%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">In vitro selection of signaling aptamers.</a> Nat Biotechnol. 18 (12), 1293-1297 (2000).</li>
<li>Khavrutskii, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+purification-free+method+of+binding+affinity+determination+by+microscale+thermophoresis%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Protein purification-free method of binding affinity determination by microscale thermophoresis.</a> J Vis Exp. (78), (2013).</li>
<li>Ramakrishnan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Probing+cocaine-antibody+interactions+in+buffer+and+human+serum%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Probing cocaine-antibody interactions in buffer and human serum.</a> PLoS One. 7 (7), 40518(2012).</li>
<li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antiviral+activity+and+interaction+mechanisms+study+of+novel+glucopyranoside+derivatives%5BTitle%5D)+AND+%22Bioorg+Med+Chem+Lett%22%5BJournal%5D)">Antiviral activity and interaction mechanisms study of novel glucopyranoside derivatives.</a> Bioorg Med Chem Lett. 25 (18), 3840-3844 (2015).</li>
<li>Wan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+into+the+molecular+recognition+of+the+granuphilin+C2A+domain+with+PI%284%2C5%29P2%5BTitle%5D)+AND+%22Chem+Phys+Lipids%22%5BJournal%5D)">Insights into the molecular recognition of the granuphilin C2A domain with PI(4,5)P2.</a> Chem Phys Lipids. 186 (4,5), 61-67 (2015).</li>
<li>Harazi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Interaction+of+UDP-N-Acetylglucosamine+2-Epimerase%2FN-Acetylmannosamine+Kinase+%28GNE%29+and+Alpha-Actinin+2+Is+Altered+in+GNE+Myopathy+M743T+Mutant%5BTitle%5D)+AND+%22Mol+Neurobiol%22%5BJournal%5D)">The Interaction of UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) and Alpha-Actinin 2 Is Altered in GNE Myopathy M743T Mutant.</a> Mol Neurobiol. , (2016).</li>
</ol>

C.E. and T.S. are employees of 2bind GmbH, which provides biophysical analytical services. &#160;Publication fees for this video-article are paid for by 2bind GmbH.

Quality controls:
Unspecific sticking/adsorption of sample material to surfaces, as well as aggregation effects, have a dramatic influence on the quality of the affinity data. However, only a few state-of-the-art technologies offer accurate and rapid options to monitor and avoid these effects. MST offers integrated quality controls that detect and help to overcome these issues, allowing for the stepwise optimization of the technical setup. Important information on sticking and fluorescence effect...

Interaction between molecules is the basis of nature. Hence, scientists in many fields of basic and applied research try to understand the fundamental principles of molecular interactions of different kinds. MicroScale Thermophoresis (MST) enables scientists to perform the fast, precise, cost-efficient, and quality-controlled characterization of molecular interactions in solution, with a free choice of buffers. There are already more than 1,000 publications using MST, from 2016 alone, describing different kinds of analyses, including library screenings, binding event validations, competition assays, and experiments with multiple binding partners1-8. In general, MST permits the study of the classical binding parameters, such as binding affinity (pM to mM), stoichiometry, and thermodynamics, of any kind of molecular interaction. A great advantage of MST is the ability to study binding events independent of the size of the interaction partners. Even challenging interactions between small nucleic acid aptamers (15-30 nt) and targets such as small molecules, drugs, antibiotics, or metabolites can be quantified.
Current state-of-the-art technologies to characterize aptamer-target interactions are either lab-intense and highly complex or fail to quantify aptamer-small molecule interactions9,10. Surface Plasmon Resonance (SPR)-based assays11,12 and truly label-free calorimetric approaches, such as Isothermal Titration Calorimetry (ITC)13-15, isocratic elution16, equilibrium ﬁltration17,18, in-line probing19, gel-shift assays, stopped-ﬂow ﬂuorescence spectroscopy20,21, fluorescence anisotropy (FA)22,23, single-molecule ﬂuorescence imaging24,25, and Bio-layer interferometry (BLI)26 are also either imprecise or incompatible with aptamer-small molecule interactions. Other principal issues of these methods are low sensitivity, high sample consumption, immobilization, mass transport limitations on surfaces, and/or buffer restrictions. Only a few of these technologies provide integrated controls for aggregation and adsorption effects.
MST represents a powerful tool for scientists to overcome this limitation to study the interactions between aptamers and small molecules27-29, as well as other targets such as proteins30-33. The technology relies on the movement of molecules through temperature gradients. This directed movement, called "thermophoresis," depends on the size, charge, and hydration shell of the molecule34,35. The binding of a ligand to the molecule will directly alter at least one of these parameters, resulting in a changed thermophoretic mobility. Ligands with small sizes may not have considerable impact in terms of size change from the unbound to the bound state, but they can have dramatic effects on the hydration shell and/or charge. The changes in the thermophoretic movement of molecules after interactions with the binding partner enables the quantification of basic binding parameters2,7,34,36,37.
As depicted in Figure 1A, the MST device consists of an infrared laser focused onto the sample within the glass capillaries using the same optics as for fluorescence detection. The thermophoretic movement of proteins via the intrinsic ﬂuorescence of tryptophans6 or of a fluorescently labeled interaction partner3,8 can be monitored while the laser establishes a temperature gradient (ΔT of 2-6 °C). The resulting temperature difference in space, ΔT, leads to the depletion or accumulation of molecules in the area of elevated temperature, which can be quantified by the Soret coefﬁcient (ST):
<img alt="Thermodynamic equation, \( S_T \times \frac{c_{hot}}{c_{cold}} = \exp(-S_T \Delta T) \), formula." src="/files/ftp_upload/55070/55070eq1.jpg" data-altupdatedat="1788235205000" data-alt="Equation">
chot represents the concentration in the heated region, and ccold is the concentration in the initial cold region.
As shown in Figure 1B, a typical MST experiment results in an MST movement profile (time trace), consisting of different phases, which can be separated by their respective timescales. The initial fluorescence is measured in the first 5 s in absence of the temperature gradient to define the precise starting fluorescence and to check for photobleaching or photoenhancement. The Temperature Jump (T-Jump) represents the phase in which the fluorescence changes before thermophoretic movement. This initial decrease in fluorescence depends on heat-dependent changes of ﬂuorophore quantum yield. The thermophoresis phase follows, in which the fluorescence decreases (or increases) due to the thermophoretic movement of the molecules until the steady-state distribution is reached. The reverse TJump and concomitant back diffusion of ﬂuorescent molecules can be observed as indicated in Figure 1B after the laser is switched off. In order to access basic binding parameters, different molar ratios of the interaction partners are analyzed and compared. Typically, 16 different ratios are studied in one MST experiment, whereas the optical visible molecule is kept constant and is supplied with an increasing amount of the unlabeled ligand. The interaction between the two binding partners induces changes in the thermophoresis, and thus in the normalized ﬂuorescence, Fnorm, which is calculated as following:
<img alt="Static equilibrium formula; Fnorm=F(hot)/F(cold); equations; thermodynamic analysis; physics study." src="/files/ftp_upload/55070/55070eq2.jpg" data-altupdatedat="1788235205000" data-alt="Equation">
Fhot and Fcold represent averaged ﬂuorescence intensities at deﬁned time points of the MST traces. Binding affinities (Kd or EC50 values) can be calculated by curve fitting (Figure 1C).
Overall, MST is a powerful tool to study molecular interactions of any kind. This manuscript offers a protocol to characterize the challenging interaction between the small molecule adenosine triphosphate (ATP; 0.5 kDa) and the 25-nt short ssDNA aptamer DH25.42 (7.9 kDa). Over the course of the manuscript, the binding site of the aptamer on the ATP molecule is mapped down to the adenine group of the ATP.

<table><tbody><tr><td>Aptamer binding buffer</td><td></td><td></td><td>20 mM Tris pH 7.6; 300 mM NaCl; 5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;; 0.01% Tween-20</td></tr><tr><td>Fluorescently labeled ATP aptamer</td><td>IDT, Leuven, Belgium</td><td></td><td>sequence: DH25.42 50-Cy5-CCTGGGGGAGT-&lt;br/&gt;ATTGCGGAGGAAGG-3</td></tr><tr><td>ATP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A2383</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>ADP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A2754</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>AMP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A2252</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>Adenine</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A8626</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>SAM</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A7007</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>dATP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>11934511001</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>CTP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>C1506</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>GTP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>G8877</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>Monolith NT.115&amp;nbsp;</td><td>NanoTemper Technologies, Munich, Germany</td><td>MO-G008</td><td>Blue/Red Channel&lt;br/&gt; MST device with standard detector, Monolith NT115 pico is MST device with high sensitivity detector</td></tr><tr><td>Monolith NT.115 capillaries Standard</td><td>NanoTemper Technologies, Munich, Germany</td><td>MO-K002</td><td></td></tr><tr><td>Eppendorf PCR tubes</td><td>Eppendorf, Germany</td><td>30124537</td><td></td></tr><tr><td>Monolith control software. 2.1.33, pre-installed on the device</td><td>NanoTemper Technologies, Munich, Germany</td><td></td><td></td></tr><tr><td>MO.affinity analysis v2.1.1</td><td>NanoTemper Technologies, Munich, Germany</td><td></td><td></td></tr><tr><td>Kaleidagraph 4.5.2</td><td>Synergy Software</td><td></td><td></td></tr></tbody></table>

mapping the binding site of an aptamer on atp using microscale thermophoresis

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

In this study, MST was applied to characterize the binding site of the DH25.42 DNA aptamer18 on ATP. In contrast to other studies characterizing the interaction of ATP or ATP-mimicking small molecules with proteins randomly labeled with one or more fluorophores38-40, this study includes a labeled version of the 7.9 kDa ssDNA aptamer with one Cy5 molecule on the 5´ end. Different ATP derivatives and related molecules, all differing from ATP in various positions, were ...

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

The overall goal of this experiment is to characterize DNA aptamer small molecule interactions, including binding site mapping, using microscale thermophoresis. This method can help to answer key questions on basic binding parameters of molecular interactions. The main advantage of this technology is that it is independent of interaction partner size, allowing quality control data of challenging interactions between aptamers and small molecules to be obtained.Though this method can provide insight into aptamer small molecule interactions, it can also be applied to other molecular interactions such as drug-target or antigen/antibody interactions. To begin the procedure, prepare a 100 micromolar stock solution of the Cy5 labeled DNA DH25.42 aptamer in distilled water. Then, dilute the aptamer stock solution to 200 nanomolar with binding buffer.Incubate the aptamer working solution for two minutes at 90 degrees Celsius. Cool the solution to room temperature on ice. Next, label and set out low binding 200 microliter microcentrifuge tubes for a 16-step serial dilution of the ligand to be tested.Pipette 20 microliters of a 10 millimolar stock solution of ligand and binding buffer into tube 1. Pipette 10 microliters of binding buffer into tubes 2 through 16. Then transfer 10 microliters of ligand stock from tube 1 to tube 2 and mix well with the pipette.Transfer 10 microliters of the diluted mixture to tube 3 and mix well. Continue the serial dilution in this way, correcting for buffer dilution effects if needed, and discard the excess 10 microliters from tube 16 when finished. Add 10 microliters of the aptamer working solution to each dilution and mix well with the pipette.Incubate the samples for five minutes at room temperature. Then draw each mixture into a clean standard capillary, being sure to only touch the capillary at the ends. Place the capillaries in the tray holder in descending order of concentration.Start the MST device and open the instrument control software. Enable manual temperature control and set the instrument temperature to 25 degrees Celsius. Once the instrument is at the correct temperature, insert the capillary tray.Set the LED channel to red for Cy5 labeled aptamer. Set the LED power to attain a fluorescence signal of 500 units. Then perform a capillary scan to obtain the capillary positions and check the sample quality.If the resulting peaks are U-shaped or flattened, prepare new samples in coded capillaries to minimize sticking effects. In the MST instrument software, fill in the ligand concentration and dilution type for the first capillary. Click and drag to fill in the concentrations and dilutions for the remaining capillaries.Enter the concentration of the aptamer in the final mix. Ensure that the experiment method is set to detect fluorescence for five seconds, record the MST for 30 seconds, and then record the fluorescence for five seconds after turning off the laser. Set the laser power to 20%Set the file path and save the experiment file.Then start the MST measurement. Obtain at least three measurements in this way. To begin data analysis, open the MST analysis software and load the experiment file.Select MST for the analysis type. The analysis software allows to compare technical repeats where all the measurements were of the same set of capillaries. Biological repeats from a different set of capillaries can be added to the analysis as well.Click the information button under the first measurement. Inspect the MST traces for bumps or spikes indicating aggregation or precipitation effects. Then inspect the capillary scan and capillary shape overlay for flattened or U-shaped peaks indicating sticking or absorption effects.As aggregates and absorption effects are rapidly detectable in MST, the method allows for fast and rapid optimization of the technical set-up to ensure optimal data quality. Check for ligand dependent fluorescence quenching or enhancement effects in the capillary scan and initial fluorescence. Inspect the fluorescence change over time for signs of photo bleaching or photo enhancement.Once the data quality has been verified, switch to the dose response mode. Select the expert analysis setting in the T Jump MST evaluation strategy. Then select the hill model for curve fitting to automatically calculate the binding parameters.In the compare results menu, select a normalization type. Export the normalized data as a spreadsheet or PDF file. In this procedure, MST was used to investigate the binding interactions of a single strand DNA aptamer with a selection of small molecule ligands.The MST curves were fit with the hill equation and the half maximal effective concentration values were determined. In five independent biological repeat measurements of the aptamer with ATP, the ATP aptamer interaction showed a negative binding amplitude of 6 to 13 units, and an average EC50 value of about 52.3 micromolar. The data for each ligand was normalized to the fraction of bound molecules and then compared.The EC50 values for ADP, AMP, and SAM compared to ATP suggest that the number of phosphate groups has at most a minor influence on the aptamer binding behavior. Removal of the robo C2 hydroxal group also showed only a minor effect on the aptamer binding affinity. The aptamer did not bind in the absence of a pairing group or if the pairing group was changed to guanine.The aptamer did bind to adenine alone, further indicating that the adenine group is the main binding site for the aptamer. Once mastered, this technique can provide essential information on basic binding parameters in minutes to hours if performed properly.

mapping-binding-site-an-aptamer-on-atp-using-microscale

Research

JoVE Journal

Biochemistry

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions.  These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Primer-Free Aptamer Selection Using A Random DNA Library

This video demonstrates microscale thermophoresis for studying the protein-lipid interaction. The assay detects the interaction between molecules by quantifying the thermophoretic movement of fluorescent-labeled proteins in response to a temperature gradient. The fluorescent molecule is mixed with different concentrations of the non-fluorescent lipid molecules, and the mixture of molecules in the solution is loaded into capillaries. A temperature gradient is applied to the samples in the...

Microscale Thermophoresis to Study Protein-Lipid Interactions in Solution

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as Förster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular...

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

The present protocol demonstrates the use of a fast, 3-stage, aptamer-based exponential amplification assay to detect targets. Sample preparation, signal amplification, and color development are covered to implement this system to recognize the presence of theophylline over that of caffeine.

Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction

This protocol outlines the design of split hybridization probes utilizing fluorescent light-up aptamer DAP-10-42 as a signal reporter and their application for nucleic acid detection and differentiation between targets with single-nucleotide substitutions.

Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis

Learn MicroScale Thermophoresis for mapping DNA aptamer binding sites to small molecules like ATP. This method determines affinity and stoichiometry independent of partner size.

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

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