Authors: Kady-Ann Steen, Nathan A. Siegfried & Kevin M. Weeks
SHAPE (selective 2’-hydroxyl acylation analyzed by primer extension) chemistry is a powerful approach for single-nucleotide resolution analysis of RNA secondary and tertiary structure. SHAPE uses hydroxyl-selective electrophiles to react with and form covalent adducts at the ribose 2’-hydroxyl group. The most useful and robust reagent for routine SHAPE experiments is 1-methyl-7-nitroisatoic anhydride (1M7). This protocol describes a straightforward synthesis of 1M7 from commercially available precursors. The synthesis of 1-methyl-7-nitroisatoic anhydride was originally described by Mortimer et al. . The reaction scheme is:
Amounts of each starting material can be varied using the above Table.
Yield ~80%; bright orange powdery solid.
1H NMR [400 MHz, CO(CD3)2]: σ 3.69 (s, 3H, -NCH3-), 8.12 (dd, J=8.8 Hz, 2 Hz, 1 H, ArH), 8.2 (d, J=2hz, 1h, ArH), 8.34 (d, J=8.4 Hz, 1 H, ArH).
Selective 2′-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA. Kady-Ann Steen, Nathan A Siegfried, and Kevin M Weeks. Nature Protocols 6 (11) 1683 - 1694 doi:10.1038/nprot.2011.373
Kady-Ann Steen, Nathan A. Siegfried & Kevin M. Weeks, Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290
Correspondence to: Kevin M. Weeks ([email protected])
Source: Protocol Exchange (2011) doi:10.1038/protex.2011.255. Originally published online 3 November 2011.