Fluoressence software

BSA-TMR- and DY-781 -loaded hydrogel samples were then subjected to a release study in order to evaluate their release from these hydrogel matrices. For this purpose, dried hydrogel samples were taken and placed in glass vials with conserved PBS pH 7.4 as the release medium. Glass vials were placed in a shaking water bath at 60 rpm and 37 °C temperature. The water bath was protected from the sunlight. Then, 500 μL of aliquots were taken at different time intervals and were replaced with the same volume of fresh PBS in order to maintain the sink conditions of the release media. BSA-TMR and DY-781 aliquots were then analyzed through fluorescence spectroscopy by using a FluoroMax-4 spectrofluorometer (HORIBA Jobin Yvon GmbH, Bensheim, Germany). The detection of the DY-781 signal involved a single-point acquisition with an excitation wavelength of 784 nm and an emission wavelength of 796 nm, while for BSA-TMR the excitation wavelength was 535 nm and the emission length was 576 nm. Measurements were conducted in a 10 mm quartz cuvette, and the data obtained were analyzed using FluorEssence™ software (HORIBA Jobin Yvon GmbH, Version 3.8.0.60). The final calculation of the release data was performed by constructing a calibration curve of BSA-TMR and DY-781. The experiment was performed in triplicate.

0.5 μM FAM-1092-S was incubated with chaperones at the indicated concentrations for 45 min. HSC70 was incubated in buffer G, DJA1 and DJA2 were incubated in buffer H. The fluorescence signal for each condition was measured at 492 nm wavelength excitation, 520 nm wavelength emission, and 5 nm slit width, using a Horiba FluoroMax spectrofluorometer. Data were collected with the FluorEssence software (Horiba). Dissociation constants (K_d) were determined by nonlinear regression fit of the averaged data to a one site – total binding model, using GraphPad Prism 8. The 95% confidence intervals and R² correlation coefficients are reported (Supplemental Table S3).

The equilibrium binding of RBM7 RRM to RNA was characterized by fluorescence anisotropy. The RNA was labeled at 5′ end with fluorescein fluorophore. The fluorescein was excited at 488 nm and its emission was collected at 520 nm. The width of both excitation and emission monochromatic slits was varying from 9 to 14 nm depending on measured RNA sequence. Integration time was set to 3 s. All measurements were conducted on a FluoroMax-4 spectrofluorometer (Horiba Jobin-Yvon). The instrument was equipped with a heated cell holder with a Neslab RTE7 water bath (Thermo Scientific). The system was operated by FluorEssence software (version 2.5.3.0 and V3.5, Horiba Jobin-Yvon). All measurements were performed at 20°C in 50 mM Tris 7.0 buffer supplemented with 200 mM sodium chloride and 10 mM 2-mercaptoethanol (pH 8). Ten nanomoles of RNA (in volume of 1.4 ml) was titrated with increasing amounts of RBM7 RRM protein sample (in the same buffer). Each data point in plot is an average of three measurements. The data were analyzed using Gnuplot (version 4.4.3) and Xmgrace (version 5.1.16). The data were normalized for visualization purposes and the experimental isotherms were fit to a single-site binding model according to Heyduk and Lee using non-linear least squares regression.