Maren Butz & Sean Devenish
The interactions of proteins with secondary molecules are of key importance in biology. Here we show how microfluidic diffusional sizing (MDS) can be used to measure KD experimentally, with no calibration or specialist preparation required. The KD of thrombin interacting with two different aptamers is successfully assessed by this method, with the values obtained in good agreement with those previously reported.
The interactions of proteins with secondary molecules is of great importance across the life sciences.
These interactions may reflect a protein-protein interaction, for example in signal transduction, an interaction between a protein and a non-protein molecule such as a drug binding its target, or a small molecule binding to change a protein’s function or stability. Whatever the reason, proper characterization of the dissociation constant, KD, is integral to understanding the interaction.
While methods exist to determine KD experimentally, reviews remark that these often require a degree of expertise to collect reliable data (1) and can have technique specific limitations – e.g. immobilizing a protein for surface plasmon resonance can render it inactive, Co-IP can see significant background noise unless appropriately specific antibodies are used, and spectroscopic techniques require prior knowledge of the expected change upon binding (be it fluorescence, NMR shift, circular dichroism etc) (2).
Here we present a new technique to characterize protein interactions in vitro; microfluidic diffusional sizing (MDS). Crucially this technique is extremely simple and rapid – with no calibration, specialist preparation or setup required. After titrating the two species (one pre-labelled) the average size of the labelled species is measured and is quickly related back to KD by standard fitting.
In this work the binding of serine protease thrombin to two aptamers (HD22 and TBA) is assessed. The KD values determined for each interaction are found to be in good agreement with previously reported values.
250 NIH units of lyophilized Thrombin (Sigma Aldrich, product code T1063) was reconstituted in 100 µL purified H2O. The concentration was verified to be 600 µg/mL (= 16.3 µM) by testing on a Fluidity One.
Two aptamers were separately assessed for their binding to thrombin; HD22 and TBA. Each were purchased pre-labelled with Alexa Fluor 647 – full details in appendix.
For each species, 100 µM aptamer in H2O was diluted to a final concentration of 1 nM aptamer in 50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM MgCl2, 0.1% BSA for the experiment.
Sample preparation for titration
A set of samples were prepared to assess the KD of each aptamer binding thrombin. For these the concentration of aptamer was held constant at 1 nM in each sample, while the concentration of thrombin was varied from 0 nM to 1630 nM, to reflect values above and below the expected KD. Full details are given in the appendix.
All samples were prepared at the same time and then tested in order from lowest to highest thrombin content. Samples were stored at 4 °c between preparation and testing.
To test each sample a 5 µL aliquot was pipetted onto a microfluidic chip and tested using a Fluidity One-W instrument. This reports the average hydrodynamic radius (Rh) of the labelled species following microfluidic diffusional sizing (MDS) (3). In this instance it means that the average size of unbound and bound aptamers in any sample was reported. Hence we observe an overall change from the Rh of the aptamer alone (with 0 µM thrombin added), to the Rh of the bound aptamer-thrombin complex (when an excess of thrombin is added to ensure the KD is exceeded).
Each sample was tested in triplicate, and the average values were fitted to a standard binding equation – see Figure 1. Fit parameters are shown in Table 1. The KD results obtained are in good agreement with literature values.
Figure 1: Results from the titration of HD22 and TBA with Thrombin. Full breakdown of results and the fitting equation used are given in Appendix 1.
Table 1: Comparison of the measured KD found in this work and the literature reported KD values. The fit parameters are also shown; Rh-free , the hydrodynamic radius of the free aptamer, and Rh-complex , the hydrodynamic radius of the aptamer-thrombin complex.
Two pre-labelled aptamers were mixed with an unlabelled protein and using microfluidic diffusional sizing (MDS) on a Fludity One-W the KD of each interaction was successfully calculated. The calculated KD values are in good agreement with previously reported values, and the difference in binding affinity is clearly visible.
The technique presents a simple means to experimentally determine KD, and crucially does so without the need to alter the molecules beyond addition of a standard fluorescent label to one binding partner. Measuring size gives confirmation of on-target binding, by quick comparison of the expected and observed size. The method has no surface fixing, and no complex preparation steps—allowing binding to be observed rapidly and in near-native conditions.
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