Melt curve analysis

To determine if the mutations introduced in tRNA^Sec affected tRNA structure and fold, a melt curve analysis was performed. For the melt curve analysis, purified tRNA^Sec samples were first treated with DNase to ensure there was no residual DNA carried over from the transcription reaction. All buffers were prepared with DEPC-treated water to ensure inhibition of RNase activity. For DNase treatment, 10 µg of each purified tRNA was resuspended in 100 µL of 1× DNase I reaction buffer (10 mM Tris–HCl, pH 7.5, 2.5 mM MgCl₂, and 0.1 mM CaCl₂) with a final DNase I (Thermo Fisher) concentration of 1 U/µL. The reaction was incubated at +37°C for 10 min. Subsequently, the RNA was purified using a RNA Clean & Concentrator kit (Zymo Research) and eluted in a buffer containing 20 mM Tris–HCl, pH 7.5, and 50 mM NaCl. For RNase A treatment of WT tRNA^Sec, a 50 µL reaction containing 500 ng/µL of WT tRNA^Sec was incubated with RNase A (100 µg/mL) in 20 mM Tris–HCl, pH 7.5, and 50 mM NaCl, at +37°C for 10 min. For tracking RNA denaturation, a 1:20,000 SYBR Green I Nucleic Acid Gel Stain (Thermo Fisher) dilution gave a superior signal-to-noise level. Practically, a 10× SYBR Green I Buffer was prepared in 100 mM Tris–HCl, pH 7.5, 20 mM MgCl₂, 1.5% (v/v) Triton X-100, and 1.5 M NaCl by diluting the original dye 2000-fold. This solution could be stored at +4°C for up to a week. To prepare samples for the melt curve analysis, each tRNA sample (no RNA, WT tRNA^Sec, mutants 1-10, and RNase A-treated WT tRNA^Sec) was diluted in the 1× SYBR Green I Buffer to a concentration of 60 ng/µL. The melt curve analysis was performed in triplicate using a 20 µL sample volume on a Bio-Rad CFX Connect instrument with a temperature gradient of 0.5°C/s from +50 to +95°C. The T_m was best visualized as a clear peak in the negative first derivative of the melting curve (−dRFU/dT).