Alexa fluor 546 c5 maleimide

Texas Red C2 Maleimide (Thermo Fisher Scientific), Oregon Green® 488 Maleimide (Thermo Fisher Scientific), Alexa Fluor 488 C5 Maleimide (Thermo Fisher Scientific), Alexa Fluor 546 C5 Maleimide (Thermo Fisher Scientific) and BADAN (Thermo Fisher Scientific) were used for specific labelling of the cysteine mutants. Proteins were incubated with a fivefold molar excess of the dye while gently shaking overnight at 4 °C. Unreacted dye was removed from the labeled protein with a PD-10 desalting column (GE Healthcare) using HP150 buffer with 0.1 mM TCEP.

RNA oligonucleotides were labelled with Alexa Fluor 546 C5-maleimide (Thermo Fisher Scientific, Waltham, MA, USA) using the 5′ EndTag Nucleic Acid Labelling System (Vector Laboratories, Burlingame, CA, USA). Labelled RNAs were purified using a denaturing polyacrylamide gel and precipitated with ethanol in the presence of 20 µg glycogen. Five pmol labelled RNAs in a transcription-buffer (T-buffer) containing 50 mM HEPES-KOH (pH 7.6), 5 mM magnesium acetate, 100 mM potassium glutamate, 2 mM spermidine and 0.01% (v/v) Tween 20 were incubated at 70 °C for 5 min and cooled to 37 °C at a rate of 1 °C min⁻¹. The samples were incubated with 0.02 U of RNase T1 (Roche, Basel, Switzerland) at 37 °C for 10 min and electrophoresed on a 20% denaturing polyacrylamide gel at 70 °C. The fluorescence signal in the gel was imaged using a FLA-5100 fluorescence image scanner (Fuji Film, Tokyo, Japan) with 532 nm excitation and 575 nm emission.