1-Methyl-7-nitroisatoic anhydride

Guide (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
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.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.