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

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.