OPTN RNA Binding Dynamics

One proposed function of OPTN is to bind RNA.^{35 (link)} A specific RNA sequence 5′ UGUGUGUGUGUGUGUGUGUGUGUG 3′, was custom synthesized based on a consensus sequence to which RNA-binding proteins like FUS and TDP43 binds.^{41 (link)} A stock solution of 100 μM RNA was prepared in nuclease-free water. OPTN was taken at 7.5 μM concentration in a 1 cm quartz cuvette as the analyte and titrated against increasing concentrations of RNA from 0.5–15 μM, till saturation was reached. Following excitation at 295 nm, the fluorescence emission for each sample was recorded from 310 nm to 400 nm. Tryptophan fluorescence quenching of OPTN in the presence of RNA was analysed through the Stern–Volmer equation;^{42 (link)} where F₀ and F are the tryptophan fluorescence intensities in the presence and absence of RNA (quencher), respectively, [Q] is the concentration of RNA, and K_SV is the Stern–Volmer quenching constant.
To analyse the binding interaction between OPTN and RNA, the binding constant was determined from the static quenching interaction. The following equation^{42 (link)} was used to determine the equilibrium between the free and bound species; where K is the binding constant and n is the number of binding sites.
To assess RNA binding with denatured OPTN, the fluorescence titration experiments were simultaneously performed at 60 °C.
To assess the functional reversibility of OPTN from a thermally denatured state, the heated and cooled protein was again titrated with an increasing concentration of RNA, and the binding constants in each case were determined as mentioned above.