Dissecting RNA folding by nucleotide analog interference mapping (NAIM)

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Nucleotide analog interference mapping (NAIM) is a chemogenetic technique.
• It enables atomic-level characterization of RNA structure and function.
• NAIM involves incorporating nucleotide analog phosphorothioates into RNA transcripts.

Purpose of the Study:

• To describe a protocol for setting up a NAIM assay for RNA folding studies.
• To provide a method adaptable for studying various RNAs and their properties.
• To detail the identification of RNA functional groups critical for specific processes.

Main Methods:

• Incorporation of random nucleotide analog phosphorothioate modifications into RNA.
• Selection of RNA based on its ability to perform a specific task (e.g., folding).
• Isolation of functional and non-functional RNA, followed by mapping modified nucleotide positions using iodine cleavage of phosphorothioate linkages.

Main Results:

• Identification of RNA functional groups essential for folding and other processes.
• Demonstration of NAIM's utility in analyzing RNA structure, catalysis, and ligand interactions.
• The protocol is adaptable for diverse RNA studies.

Conclusions:

• NAIM is a powerful tool for detailed RNA analysis.
• The described protocol facilitates the study of RNA folding and other functions.
• Reliable results require several weeks, depending on RNA length and modification number.