The principle of mismatch DNA-binding assay by his-tagged MutS immobilized on a nickel-coated microplate is presented in Fig. 1. In brief, the his-tagged MutS protein is loaded to microplate wells to become immobilized after short incubation. The wells are emptied and washed to remove the unbound protein. For protein–DNA binding, the examined, biotinylated DNA fragments are added to the wells. After short incubation, the wells are emptied and washed in order to remove the unbound DNA. The captured DNA is detected with horseradish peroxidase conjugated to avidin. Avidin binds biotin labels of captured DNA, while horseradish peroxidase catalyses the oxidation of TMB chromogenic substrate, thus developing blue-green colour. The results could be quantitated with an ELISA reader.
In order to examine mismatch DNA-binding properties of MutS, we compared the effects of capturing mismatched and fully complementary DNA. We designed an experimental setup where there are three microplate wells with immobilized his-tagged MutS. The mismatched DNA is added to the first well, while the fully complementary DNA and the loading buffer without DNA are loaded to the second and third control wells, respectively. MutS-DNA-binding affinity is estimated as the absorbances obtained in the colorimetric assay. The specificity towards the mismatch is calculated as the ratio of the signal for mismatched DNA to that for fully complementary DNA.
DNA Mismatch Binding Affinity and Specificity at Different Binding Conditions
In order to find the conditions where MutS proteins show high affinity for DNA mismatches and low non-specific binding to fully complementary DNA, we tested the impact of salt concentration and ADP presence in the buffer used for DNA binding and washing. We contrasted the results obtained for the buffer containing 150 mM salt (Figs. 2a, 3a) with those obtained for the buffer with doubled (300 mM) salt concentration (Fig. 2b), and for the 300 mM salt buffer with 1 mM ADP addition (Fig. 2c). We performed the experiments with three different his-tagged MutS proteins from Escherichia coli, Deinococcus radiodurans, and Thermus thermophilus. The proteins used in the experiments were purified from recombinant overproducing E. coli strains, and the his-tag tails were introduced as the translational fusions at their N-termini.
As a result, we were able to find that under 300 mM salt concentration the immobilized his-tagged MutS proteins exhibit both efficient binding of mismatched DNA and satisfactory specificity. Further, we observed that under the conditions of experiment, MutS from D. radiodurans showed a higher affinity towards mismatched DNA than those two others (Fig. 2a, b). In the presence of ADP, the specificity of MutS from T. thermophilus strongly increased (Fig. 3b) to exceed even that of MutS from D. radiodurans (Fig. 2c).
Impact of DNA Concentration and Protein Amount
We performed two series of experiments in which either the amount of DrMutS protein added to the wells was kept constant (1 µg), while the DNA concentrations varied in the range of 0.0001–1.0 µM (Fig. 4a) or the concentration of loaded DNA was at the constant level (0.1 µM), whereas the amounts of loaded DrMutS varied from 0.05 to 2 µg (Fig. 4b). The obtained absorbance values were correlated with increasing amounts of either added DNA (Fig. 4a) or loaded MutS protein (Fig. 4b). The series of DNA-binding tests was performed in the buffer containing 150 mM salt without ADP addition, which corresponds to the conditions of the experiment presented in Fig. 2a. The measurements showed that the absorbance changed linearly with the increasing concentrations of loaded DNA within the range 0.001–0.1 µM (Fig. 4a, inset). Also, the results indicate that the working amounts of loaded his-tagged protein correspond to the range of 1–20 pmol (Fig. 4b, inset). A slight improvement in DNA mismatch binding specificity could be observed at lower amounts of loaded DNA (Fig. 5a). Changing the amounts of loaded MutS did not have an essential impact on specificity towards mismatched DNA (Fig. 5b). However, lowering DNA concentrations results in a decrease of colorimetric signal (Fig. 4a).
Evaluation of Detection Sensitivity
The microplate assay with 1 µg of his-tagged-MutS loaded for immobilization was demonstrated to allow reliable detection of captured biotinylated DNA following binding in 100 µl volume at the concentrations ranging from 0.001 to 0.1 µM (Fig. 4a). In order to evaluate the limits of detection, the assay was performed for a series of decreasing DNA concentrations of mismatched DNA (0.01, 0.001, 0.0001 µM) and prolonged time of colorimetric reaction (Fig. 6). The results showed that the signal for 10−4 µM was close to the background level. In the case of DNA concentrations of 0.01 and 0.001 µM, the colorimetric signal could be easily discriminated from that of background. Nevertheless, we would recommend applying a higher working concentration of biotinylated DNA of 0.1 µM, where stronger signals are produced, as more useful to test different binding conditions (Fig. 2). Extending the time of colorimetric reaction from 15 to 245 min did not result in significant improvement of detection limit, while approximately a 25 % increase in signal was observed after a 45-min as compared to a 15-min reaction.