Analysis of Ribozyme Structure and Function by Nucleotide Analog Interference Mapping

Nucleotide analog interference mapping (NAIM) is a quick and effective method to define concurrently, yet singly, the importance of specific functional groups at particular nucleotide residues in relation to the structure and function of an RNA. NAIM can be utilized on virtually any RNA with an assayable function, including catalytic RNAs. The method hinges on the ability to successfully incorporate, within an RNA transcript, various 5′-
O
-(1-thio)nucleoside analogs randomly via in vitro transcription. This can be achieved by using wild-type or Y639F mutant T7 RNA polymerase, thus creating a pool of analog-doped RNAs. When subjected to a selection step to separate the active transcripts from the inactive ones, the pool helps to identify functional groups that are crucial for RNA activity. The technique can be used to study ribozyme structure and function via monitoring of cleavage or ligation reactions, or define functional groups that are critical for RNA folding, RNA-RNA interactions, and RNA interactions with proteins, metals, or other small molecules. All major classes of catalytic RNAs have been examined by NAIM. This is a generalized approach that should provide the scientific community with the tools to better understand the RNA structure-activity relationship (SAR).