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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 partners^1-8. 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 interactions^9,10. Surface Plasmon Resonance (SPR)-based assays^11,12 and truly label-free calorimetric approaches, such as Isothermal Titration Calorimetry (ITC)^13-15, isocratic elution¹⁶, equilibrium filtration^17,18, in-line probing¹⁹, gel-shift assays, stopped-flow fluorescence spectroscopy^20,21, fluorescence anisotropy (FA)^22,23, single-molecule fluorescence imaging^24,25, 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 molecules^27-29, as well as other targets such as proteins^30-33. 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 molecule^34,35. 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 parameters^2,7,34,36,37.

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 partner^3,8 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 (S_T):

c_hot represents the concentration in the heated region, and c_cold 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, F_norm, which is calculated as following:

F_hot and F_cold represent averaged fluorescence intensities at defined time points of the MST traces. Binding affinities (K_d 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.

Protokół

1. Preparation of the Aptamer Working Stock

1. Follow the manufacturer's instructions and dissolve the oligonucleotide (5-Cy5-CCTG GGGGAGTATTGCGGAGGAAGG-3, sequence from reference¹⁸) in water, reaching a 100-µM final concentration.
2. 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).
3. Incubate the mixture for 2 min at 90 °C, let the sample immediately cool down on ice, and use the sample at room temperature.

2. Preparation of the Ligand Dilution Series

1. 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 µl 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.
2. 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.
3. Fill 20 µl of the ligand stock (10 mM) in tube 1. Add 10 µl of aptamer binding buffer into micro reaction tubes 2 to 16.
4. Transfer 10 µl of tube 1 to tube 2 and mix properly by pipetting up and down several times. Transfer 10 µL to the next tube and repeat this dilution for the remaining tubes.
5. Discard the 10 µl excess from the last tube. Avoid any buffer dilution effects. The buffer in tube 1 and in tubes 2-16 must be identical.

3. Preparation of the Final Reaction Mix

1. Prepare the individual binding reactions with a volume of 20 µl (10 µl of aptamer working solution + 10 µl of the respective ligand dilution) to minimize pipetting errors. A volume of only 4 µl is sufficient to fill the capillary.
2. Add 10 µl of the 200 nM aptamer working solution to 10 µl of each ligand dilution and mix properly by pipetting up and down several times.
3. Incubate the samples for 5 min at room temperature and fill 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.
4. Place the capillaries onto the capillary tray and start the MST device.

4. Starting the MST Device

NOTE: The device provides two pre-installed software packages, the ''control" software for the technical setup of the experimental conditions and the ''analysis" software for the interpretation of the produced data.

1. Before placing the capillary tray into the MST device, start the control software and adjust the overall desired temperature by selecting ''enable manual temperature control" in the ''temperature control" dropdown menu. Adjust the temperature to 25 °C in this way.
   NOTE: The MST instruments can be temperature-controlled from 22 to 45 °C.
2. Wait for the temperature to reach the expected level and then place the capillary tray into the MST device.
3. Set the LED channel to ''red" for Cy5 dyes and adjust the LED power to gain a fluorescence 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.

5. Capillary Scan

1. Carry out a capillary scan to check different quality aspects of the sample by choosing the capillary position on the "control" software and clicking on "start cap scan" before starting the MST measurement.
2. 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.

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 fluorescence signal is sufficient. For more details, see the discussion.

1. Assign the ligand concentrations from the dilution series to the respective capillary position in the ''control" software. Consider the dilution step of mixing the aptamer and ligand (1:1).
2. 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.
3. Enter the concentration of the fluorescent aptamer (here, 100 nM) in the respective section of the control software.
4. Use the default settings, which detect the fluorescence 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.
5. 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.
6. Save the experiment after selecting the destination folder and start the MST measurement by pressing the ''Start MST measurement" button.
   NOTE: The .ntp file will be generated in the destination folder. Using this setup, one measurement lasts 10-15 min.
7. 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).

7. MST Data Analysis

NOTE: The analysis software enables the analysis of data on the fly during the measurement. The analysis software plots the MST time traces and changes in the normalized fluorescence (F_norm) versus the ligand concentration³⁷.

1. Start the MST analysis software (MO.Affinity Analysis) and load the .ntp file from the destination folder. Select "MST" 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.
2. Add the respective technical or biological run(s) to a new analysis by drag-and-drop or by pressing the "+" button below the respective experimental run.
3. 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.
4. 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.
5. 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.
6. Switch to the dose-response mode and change the analysis setting to "expert" mode by pressing the respective button. Select "T-Jump" as the MST evaluation strategy.
7. Select the "Hill" model for curve fitting. The binding parameters will automatically be calculated. Normalize the data by choosing the respective type of normalization in the "compare results" menu. Export the data either as an .xls or .pdf.
   NOTE: The table below the binding graph summarizes the calculated binding parameters.

Wyniki

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 fluorophores^38-40, 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, ...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Aptamer binding buffer			20 mM Tris pH 7.6; 300 mM NaCl; 5 mM MgCl2; 0.01% Tween-20
Fluorescently labeled ATP aptamer	IDT, Leuven, Belgium		sequence: DH25.42 50-Cy5-CCTGGGGGAGT- ATTGCGGAGGAAGG-3
ATP	Sigma Aldrich, Germany	A2383	10 mM stock solutions stored at - 20 °C
ADP	Sigma Aldrich, Germany	A2754	10 mM stock solutions stored at - 20 °C
AMP	Sigma Aldrich, Germany	A2252	10 mM stock solutions stored at - 20 °C
Adenine	Sigma Aldrich, Germany	A8626	10 mM stock solutions stored at - 20 °C
SAM	Sigma Aldrich, Germany	A7007	10 mM stock solutions stored at - 20 °C
dATP	Sigma Aldrich, Germany	11934511001	10 mM stock solutions stored at - 20 °C
CTP	Sigma Aldrich, Germany	C1506	10 mM stock solutions stored at - 20 °C
GTP	Sigma Aldrich, Germany	G8877	10 mM stock solutions stored at - 20 °C
Monolith NT.115	NanoTemper Technologies, Munich, Germany	MO-G008	Blue/Red Channel MST device with standard detector, Monolith NT115 pico is MST device with high sensitivity detector
Monolith NT.115 capillaries Standard	NanoTemper Technologies, Munich, Germany	MO-K002
Eppendorf PCR tubes	Eppendorf, Germany	30124537
Monolith control software. 2.1.33, pre-installed on the device	NanoTemper Technologies, Munich, Germany
MO.affinity analysis v2.1.1	NanoTemper Technologies, Munich, Germany
Kaleidagraph 4.5.2	Synergy Software