Authors: Kady-Ann Steen, Nathan A. Siegfried & Kevin M. Weeks ### Abstract 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*. ### Reagents 1. 4-nitroisatoic anhydride (4NIA) (AstaTech, cat. no. 69441) - Sodium hydride (NaH) – 60% dispersion in mineral oil (Sigma-Aldrich, cat. no. 452912) - Methyl iodide (MeI) (Sigma-Aldrich, cat. no. 289566) - Anhydrous dimethylfluoride (DMF) (Sigma-Aldrich, cat. no. 227056) - HCl (Fisher Scientific, cat. no. A144) - H2O - Ether (Fisher Scientific, cat. no. E138) **REAGENT SETUP** - H2O and HCl should be ice cold before use. ### Equipment 1. Round bottom flasks (250 and 500 mL) - Bunsen burner - N2 tank and gas line - Teflon-coated stirring bar - Rubber septa - Disposable syringes (1 mL, 20 mL, 60 mL) - Disposable needles (18 gauge) - Stirring plate - Buchner funnel - Vacuum rotary evaporator - Watch glass - Oven maintained at 90 °C **EQUIPMENT SETUP** - All glassware used in the described reactions are washed with soap and water, rinsed with water, rinsed with acetone, and flame-dried over Bunsen burner. Rubber septa are immediately placed over flasks, flasks are flushed with N2, and allowed to cool. ### Procedure 1. Dissolve 4NIA in 60 mL DMF in a 250 mL flame-dried round bottom flask under N2. - In a separate 500 mL flame-dried round bottom flask under N2, make a slurry of NaH in 15 mL DMF and stir. - Slowly add 4NIA solution dropwise to slurry of NaH in DMF. Stir for 5 minutes. - Slowly add MeI dropwise and then stir at room temperature for 4 hours. - Pour reaction into 100 mL *ice cold* 1N HCl. - Filter resulting bright orange precipitate by vacuum filtration in a Buchner funnel. - Rinse precipitate twice with *ice cold* water, followed by 3 rinses with ether. - Dry overnight in oven on watch glass. ### Anticipated Results **Analytical data** 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). ### Associated Publications **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](http://dx.doi.org/10.1038/nprot.2011.373) ### Author information **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 (weeks@unc.edu) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2177) (2011) doi:10.1038/protex.2011.255. Originally published online 3 November 2011*.