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>

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 &#64257;ltration17,18, in-line probing19, gel-shift assays, stopped-&#64258;ow &#64258;uorescence spectroscopy20,21, fluorescence anisotropy (FA)22,23, single-molecule &#64258;uorescence 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 &#34;thermophoresis,&#34; 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 &#64258;uorescence of tryptophans6 or of a fluorescently labeled interaction partner3,8 can be monitored while the laser establishes a temperature gradient (&#916;T of 2-6 &#176;C). The resulting temperature difference in space, &#916;T, leads to the depletion or accumulation of molecules in the area of elevated temperature, which can be quantified by the Soret coef&#64257;cient (ST):
<img alt="Equation" src="/files/ftp_upload/55070/55070eq1.jpg" />
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 &#64258;uorophore 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 &#64258;uorescent 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 &#64258;uorescence, Fnorm, which is calculated as following:
<img alt="Equation" src="/files/ftp_upload/55070/55070eq2.jpg" />
Fhot and Fcold represent averaged &#64258;uorescence intensities at de&#64257;ned 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

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&#180; end. Different ATP derivatives and related molecules, all differing from ATP in various positions, ...

Watch this Scientific Journal Video about Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis at JoVE.com

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

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 ...

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

Research

JoVE Journal

Biochemistry

10.5K Views.  2bind GmbH. MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

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

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

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

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 esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

Chemistry

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

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.

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

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

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

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

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

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 protein.&#160;

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

Cancer Research

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

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.

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

Biology

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

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

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.

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...

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

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules with signal-generation capability. For the system described herein, target recognition through an aptameric domain, Stem II of a modified hammerhead ribozyme, activates the self-cleaving ribozyme by stabilizing the initially unstructured construct. The cis-cleaving RNA acts at the junction of Stem III and Stem I, creating two cleavage products. The longer cleavage product primes an isothermal exponential amplification reaction (EXPAR) of the two similar catalytically active G-quadruplexes. Those resulting amplification products catalyze peroxidase reduction, which is coupled to the reduction of a colorimetric substrate with an output that the naked eye can detect. The 3-part system described in the present study improves detection modalities such as enzyme-linked immunosorbent assays (ELISAs) by producing a visually detectable signal for indicating the presence of as low as 0.5 &#181;M theophylline in as little as 15 min.

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.

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules ...

Bioengineering

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

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

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.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

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

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

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.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Differential Scanning Calorimetry to Study DNA Aptamer-Thermolabile Ligand Interaction

Source: Mittermaier, A. K., et al. <a href="https://app.jove.com/v/55959">Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions.</a> J. Vis. Exp. (2017)
This video demonstrates differential scanning calorimetry &#8212; a technique to study the interaction between a biomolecule and a ligand. The binding of a DNA aptamer and a thermolabile ligand is studied by applying repeated heating and cooling cycles to the sample and obtaining scans for thermodynamic analysis.

Różnicowa kalorymetria skaningowa do badania interakcji DNA aptamer-termolabilny ligand

Source: Mittermaier, A. K., et al. Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions. ...