Nucleotide analog interference mapping

Abstract

Single-atom substitution experiments provide atomic resolution biochemical information concerning RNA structure and function. Traditionally, these experiments are performed using chimeric RNAs generated by reassembly of full-length RNA from a synthetic substituted oligonucleotide and a truncated RNA transcript. Unfortunately, this technique is limited by the technical difficulty of assembling and measuring the effect of each singly substituted molecule in a given RNA. Here we review an alternate method for rapidly screening the effect of chemical group substitutions on RNA function. Nucleotide analog interference mapping is a chemogenetic approach that utilizes a series 5'-O-(1-thio)-nucleoside analog triphosphates to simultaneously, yet individually, probe the contribution of a functional group at every nucleotide position in an RNA molecule. A population of randomly substituted RNAs is prepared by including phosphorothioate-tagged nucleotide analogs in an in vitro transcription reaction. The active molecules in the RNA population are selected by an activity assay, and the location of the analog substitution detrimental to activity is identified by cleavage at the phosphorothioate tag with iodine and resolution of the cleavage fragments by gel electrophoresis. This method, which is as easy as RNA sequencing, is applicable to any RNA that can be transcribed in vitro and has an assayable function. Here we describe protocols for the synthesis of phosphorothioate-tagged analogs and their incorporation into RNA transcripts. The incorporation properties and unique biochemical signatures of each individual analog are discussed.