INTERFERENCE MAPPING

Nucleotide Analog Interference Mapping (NAIM) is a chemogenetic approach that makes it possible to simultaneously, yet individually, probe the contribution of a particular functional group at almost every RNA nucleotide position in a single experiment¹. The method utilizes a series of 5’-O-(1-thio)nucleoside analog triphosphates in a modification interference procedure that is as simple as RNA sequencing. In a NAIM experiment the smallest mutable unit is not the base pair, but rather the individual functional groups that comprise the nucleotides. Because the modification or deletion of a particular functional group within an RNA can severely affect its activity, this approach makes it possible to efficiently determine the chemical basis of RNA structure and function.

Instead of synthesizing a series of RNAs with chemical substitutions at specific sites, NAIM utilizes a combinatorial approach. Each nucleotide analog is prepared as a triphosphate for incorporation into the RNA during DNA templated in vitro transcription. The nucleotide analogs used in NAIM include a specific chemical alternation to the base or sugar, and an α-phosphorothioate substitution which serves as a chemical tag. The nucleotide analog triphosphate is randomly incorporated into an RNA transcript, where the phosphorothioate linkage can be selectively cleaved by the addition of I₂ to produce a series of RNA cleavage products whose lengths correspond to the sites of analog incorporation². By radioactively or fluorescently tagging one end of the RNA transcript, cleaving the RNA with I₂ , and resolving the cleavage products on a denaturing polyacrylamide gel, the sites of analog incorporation throughout the RNA can be individually assayed and used for interference analysis. The phosphorothioate tagged nucleotide analogs make it possible for all of the positions in the RNA to be assayed individually for functional group modification in a single experiment.

Because the phosphorothioate chemical tag is independent of the nucleotide analog whose location it reports, NAIM is generalizable to any analog that can be incorporated into a transcript by an RNA polymerase. A typical NAIM experiment is comprised of four steps. (i) The phosphorothioate tagged nucleotide analog is randomly incorporated throughout the RNA to create a family of transcripts, each of which contains only a few substitutions. A different transcription reaction is performed for each analog. (ii) The functional RNA variants in the population are separated from the inactive transcripts. The exact nature of the activity assay is specific for the RNA being studied, but could include affinity chromatography, native gel electrophoresis, filter binding, selective radiolabeling, etc. (iii) The phosphorothioate linkages in the active and unselected RNA populations are cleaved by I₂ addition to mark the sites of analog incorporation within each molecule. (iv) The individual RNA fragments are resolved by gel electrophoresis and visualized by autoradiography. Sites of analog substitution that are detrimental to function are scored as gaps in the sequencing ladder among the active RNA variants. Because every position in the sequence is a unique and independent band on the sequencing gel, a single screen can define the effect a particular analog has at every incorporated position within the RNA. The approach is applicable to any RNA that can be transcribed in vitro and has an assayable function that can be used to distinguish active and inactive variants. RNA functions that are amenable to this approach include catalysis, folding, protein or ligand binding, and the ability to act as a reaction substrate.

A ANALOGS

NAIM utilizes α-phosphorothioate tagged nucleotide analogs, each of which includes an incremental chemical alteration in the base or ribose sugar. The most completely developed set of analogs are those of adenosine, for which eight different analogs have been utilized in NAIM.³Five analogs modify the nucleotide base and three modify the ribose sugar. The base analogs include purine riboside (PurαS), N6-methyladenosine (m6AαS), tubercidin (7dAαS), diaminopurine riboside (DAPαS), and 2-aminopurine riboside (2APαS). The ribose sugar analogs all modify the 2'-OH group and include 2'-deoxyadenosine (dAαS), 2'-deoxy-2'-fluoroadenosine (FAαS), and 2'-O-methyladenosine (OMeAαS). All of the analogs can be randomly incorporated into an RNA transcript at an ideal 5% level of efficiency using either the wild-type T7 RNA polymerase or a Y639F RNA polymerase point mutant⁴. Each of these analogs provides specific information about the chemical basis of RNA activity at almost every incorporated position in the transcript.