Journal: Nature chemical biology
Article Title: Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling
Figure Lengend Snippet: Application of the epoxide-opening catalytic RNA to enrich RNAs of interest from total cellular RNA and to capture RNA-binding proteins ( a ) Transcriptional fusion of a self-labeling catalytic RNA to an RNA of interest may enable selective, covalent RNA modification in a complex biological sample. ( b ) Total RNA from HEK 293T cells was reacted with epoxide-azide 14 , followed by DBCO-TAMRA. Total RNA was analyzed by PAGE and TAMRA-modified RNAs were visualized by fluorescence imaging. Lanes 1 and 6: in vitro transcribed catalytic RNA-fused 5S rRNA containing one or three copies of the catalytic RNA, respectively, rather than cellular RNA. Lanes 2 and 3: the inactive C9-G35 mutant RNA. Lanes 4–8: 5S rRNA fused to one copy (lanes 4–5) or three copies (lanes 6–8) of the active optimized catalytic RNA. Bands at the top of the gel result from incomplete removal of excess DBCO-TAMRA probe or background labeling of cellular rRNAs/mRNAs. The complete gel is shown in Supplementary Figure 14 . ( c ) Western blot probing the presence of three known ASH1 mRNA-binding proteins (Puf6, Khd1, and She2) and one non-binding protein control (Guk1) in yeast cell lysate. Lanes 1 and 2: Lysate incubated overnight with streptavidin-coated magnetic beads only (lane 1) or pre-incubated with 5 µg of epoxide 1 -modified ASH1 -catalytic RNA (lane 2). Unbound proteins were washed away and captured proteins were eluted at 95 °C. Lane 3: Input lysate prior to incubation with beads. The complete gel is shown in Supplementary Figure 15 .
Article Snippet: The RNA (7 µL) and streptavidin (NEB; 1 µg) were incubated at room temperature for 25 minutes and then combined with gel electrophoresis loading buffer.
Techniques: RNA Binding Assay, Labeling, Modification, Polyacrylamide Gel Electrophoresis, Fluorescence, Imaging, In Vitro, Mutagenesis, Western Blot, Binding Assay, Incubation, Magnetic Beads