Circular dichroism spectroscopy

The structural changes in RNA G-quadruplexes (TERRA-6, TERRA-12, TERRA-24, abasicloopsTERRA-24) by UP1 and UP1+RGG were monitored using CD spectroscopy. CD titrations were carried out using JASCO J-715 Spectrophotometer. The wavelength scans for the RNAs were done from 325 to 225 nm. The average of three spectral scans was taken for each titration step. Baseline correction was done for each run and the data was normalized to molar ellipticity per residue. The titration experiments were performed in 1 cm path length cuvette from Hellma Analytics at 25°C at a scanning speed of 100 nm/min with a response time of 4 s. 5 μM of the RNA sample was titrated with increasing protein concentration (in the steps of 0, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM and 25 μM of proteins).

For the melting studies of the RNA G-quadruplexes, the change in ellipticity was measured at 262 nm as a function of increasing temperature. The ellipticity was monitored from 10°C to 100°C with an increase of temperature of 1°C every minute. Data points were recorded after every 0.5°C.