Comparison of SHAPE reagents for mapping RNA structures inside living cells

Analysis of cDNA truncations generated by various SHAPE electrophiles. (A) ³²P radioblot comparing in vitro and in vivo modification levels of FAI, NAI, NAI-N₃, and 1M7 on mouse U1 snRNA and 5S rRNA as measured by cDNA synthesis truncation and denaturing PAGE. Annotations of regions and flexibility as in Spitale et al. (2012). (B) Comparison of in vitro and in vivo modification levels of FAI, NAI, NAI-N₃, and 1M7 on Escherichia coli 5S rRNA as measured by cDNA synthesis truncation and denaturing PAGE. Red arrows denote highly conserved accessible nucleotides between mouse and E. coli in vivo SHAPE experiments (Spitale et al. 2012). (C) Comparison of in vivo modification levels of NAI-N₃ and 1M7 on intact and permeabilized mouse cells as measured by cDNA synthesis truncation of the 5S rRNA and denaturing PAGE.