Nucleotide Analog Interference Mapping

Abstract

Methods collectively known as modification interference are exceptionally powerful approaches used to identify functionally important chemical groups in the phosphodiester backbone or nucleobases of an RNA. In a modification interference assay, end-labeled RNAs that have been modified at different positions are allowed to participate in a reaction of interest, and then functional RNA molecules (e.g., those bound by protein or that successfully participate in a processing reaction) are separated from nonfunctional RNA molecules (e.g., those not bound by protein or unable to participate in a processing reaction). Nucleotide analog interference mapping (NAIM) involves the incorporation of α-thionucleotides containing a modified base into the RNA molecule of interest. The sites containing the modified base are identified by cleavage with iodoethanol. NAIM is useful whenever the thiophosphate substitution on its own does not prevent or inhibit a specific reaction. To perform NAIM, it is first necessary to perform a thiophosphate interference analysis. Any positions that are not affected by thiophosphate substitution can then be analyzed by NAIM.