Determining eIF4E-capped RNA Binding Kinetics

To determine the dissociation constant for eIF4E protein and capped short RNA complexes, an MST-based method was applied, previously described in (28 (link)), with modification of the utilized ligand form; here, capped short RNAs were analysed. Binding assay mixtures were prepared in 20 μl as follows: 10 nM fluorescent probe m⁷Gp₅OC₃(5)FAM, 50 nM murine eIF4E and ligand (differently capped short RNAs ranging from 1.75 μM to 0.05 nM) in an MST buffer (50 mM HEPES–KOH pH 7.2, 100 mM KCl, 0.5 mM EDTA, 0.05% Tween-20). Samples after preparation, without any additional incubation, were loaded into Monolith NT.115 Capillaries (MO-KO22; NanoTemper Technologies). MST was performed using a Monolith NT.115 instrument (NanoTemper Technologies) at 25°C. Instrument parameters were adjusted to 100% LED Blue power and Medium MST power. To determine the K_D,app values, a standard 1:1 binding model was fitted to the data using PALMIST software (version 1.4.4). Confidence intervals were determined using error-surface projection (ESP) (29 (link)).