1-Methyl-7-nitroisatoic anhydride

1-Methyl-7-nitroisatoic anhydride (1M7) is a reagent that detects local nucleotide flexibility, for probing 2'-hydroxyl reactivity, can be used for RNA structure analysis^[1]. In Vitro:SHAPE reaction protocol for 1-Methyl-7-nitroisatoic anhydride^[1]
1. RNA pretreatment: Incubate RNA in a buffer containing a final concentration of 10/9 for 30 minutes to allow the RNA to fully fold and reach equilibrium.
2. Prepare 1-Methyl-7-nitroisatoic anhydride modification reaction: Prepare a 1-methyl-7-nitroisatoic anhydride solution, and select the appropriate concentration based on the different RNAs (30 mM for 16S and 23S rRNA, 100 mM for RNase P RNA, and 50 mM for HIV-1 RNA). Dissolve 1-Methyl-7-nitroisatoic anhydride in DMSO to prepare a 10-fold concentrated solution.
3. Start the 1-Methyl-7-nitroisatoic anhydride modification reaction: Add 1/10 volume of 1-Methyl-7-nitroisatoic anhydride-DMSO solution to the RNA solution and mix immediately. For the non-reaction control group, only add an equal volume of pure DMSO.
4. Reaction conditions: react at 37°C for a certain time (the specific time depends on the experimental design).
5. Termination of reaction: After the reaction is completed, immediately place the reaction mixture on ice to stop the reaction.
6. RNA recovery: RNA is recovered using ethanol precipitation, the precipitate is collected by centrifugation, and the supernatant is discarded. Resuspend the RNA precipitate with a small amount of 1/23 TE buffer (5 mM Tris-HCl, 0.5 mM EDTA).
7. Analysis of SHAPE reaction products: Use primers with different fluorescent labels to analyze the modification sites of RNA by primer extension. Separate the [+] and [–] reaction products and one or two sequencing gradients by electrophoresis, and read the results by fluorescence detector.
8. Data analysis: Analyze the secondary structure information of RNA based on the changes in the intensity of the fluorescence signal.