Prior to EPR measurements, the folded labeled RNAs were first lyophilized then re-dissolved in 50/50 (v/v) glycerol/water to obtain a stock solution with the same salt concentrations as that in the SEC buffer. Control studies have shown that lyophilization does not affect global folding of the RNase P RNAs (Fig. S3† ).
For CW-EPR measurements, each EPR sample was approximately 10 μL with the spin-labeled RNA concentrations being 50–120 μM, and was loaded into a borosilicate glass capillary (1.0 mm ID × 1.2 mm OD, Fiber Optic Center, Inc.) sealed at one end. CW-EPR spectra were obtained on a Bruker EMX X-band spectrometer equipped with an ER-041X microwave Bridge and a high sensitivity cavity (ER-4119HS, Bruker Biospin, Inc). All CW-EPR spectra were acquired at room temperature with an incident microwave power of 2 mW, a modulation frequency of 100 kHz, and a modulation amplitude of 1 G. For each sample, 10 scans were collected and accumulated. The spectra were background corrected and normalized following a previously reported procedure.51 (link) Spin counting was performed based on the 2nd integral values of the baseline-corrected but not normalized CW-EPR spectra,51 (link) and the details are described in Fig. S5 and Table S5.†
For CW-EPR measurements, each EPR sample was approximately 10 μL with the spin-labeled RNA concentrations being 50–120 μM, and was loaded into a borosilicate glass capillary (1.0 mm ID × 1.2 mm OD, Fiber Optic Center, Inc.) sealed at one end. CW-EPR spectra were obtained on a Bruker EMX X-band spectrometer equipped with an ER-041X microwave Bridge and a high sensitivity cavity (ER-4119HS, Bruker Biospin, Inc). All CW-EPR spectra were acquired at room temperature with an incident microwave power of 2 mW, a modulation frequency of 100 kHz, and a modulation amplitude of 1 G. For each sample, 10 scans were collected and accumulated. The spectra were background corrected and normalized following a previously reported procedure.51 (link) Spin counting was performed based on the 2nd integral values of the baseline-corrected but not normalized CW-EPR spectra,51 (link) and the details are described in Fig. S5 and Table S5.