Extended Data Fig. 6: Self-alkylation activity of SAM riboswitches with ProSeDMA and ProSeAM.
From: Structure and catalytic activity of the SAM-utilizing ribozyme SAMURI
Streptavidin binding assay of four SAM riboswitches confirmed propargyl modification following copper-catalyzed click reaction with biotin azide. Reaction conditions: For trans- alkylation, 100 pmol riboswitch RNA, 2 nmol ProSeDMA (a) or ProSeAM (b) were incubated in 20 μL reaction buffer (50 mM HEPES, 120 mM KCl and 5 mM NaCl, 10 mM MgCl2) at 37 °C for 15 h. For click reaction, 100 pmol alkylated RNA, 500 μM CuBr, 1 mM TBTA and 1 mM biotin azide were incubated in H2O/DMSO/tBuOH = 5/3/1 solution at 37 °C for 1 h. For streptavidin gel shift assay, 5 pmol RNA and 1 μg of streptavidin were incubated in 1× TBS buffer at room temperature for 15 min. c. Concentration dependent self- alkylation of the SAM/SAH riboswitch with ProSeDMA. Same conditions were used for streptavidin binding assay. Representative gel images of independent duplicates for all riboswitches.
