Probing RNA Structure and Function by Nucleotide Analog Interference Mapping

Abstract

Nucleotide analog interference mapping (NAIM) can be used to simultaneously, yet individually, identify structurally or catalytically important functional groups within an RNA molecule. Phosphorothioate-tagged nucleotides and nucleotide analogs are randomly incorporated into an RNA of interest by in vitro transcription. The phosphorothioate tag marks the site of substitution and identifies sites at which the modification affects the structure or function of the RNA molecule. This technique has been expanded to include identification of hydrogen bonding pairs (NAIS), ionizable functional groups, metal ion ligands, and the energetics of protein binding (QNAIM). The analogs, techniques, and data analysis used in NAIM are described here.

Molecular basis for RNA kink-turn recognition by the h15.5K small RNP protein

“…ATPaS, GTPaS, CTPaS, UTPaS, PurTPaS, InoTPaS, dATPaS, dGTPaS, dCTPaS, and dUTPaS were added to transcription reactions as described (Cochrane and Strobel 2004) except for dCTPaS, which was added at 4 mM to achieve a level of incorporation comparable to the other analogs. Concentrations of unlabeled RNAs were determined spectrophotometrically.…”

Methods to Detect and Characterize Metal Ion Binding Sites in RNA

“…Therefore, a basic characterization of the RNA and the processes that are subject of the NAIM analysis are a prerequisite for the successful optimization of the selection step. Two types of selection systems have been used in the past: In the case of ribozymes, active and inactive molecules are separated based on their length difference upon either self-cleavage or ligation with a substrate [180]. If folding is the required trait, native gel electrophoresis, which separates molecules according to their overall shape, is the method of choice.…”

“…The gel solution should contain all cations that are needed for folding in the appropriate concentrations [180]. Reaction conditions are chosen, such as to yield 20 -40% of folded relative to non-folded RNA [179].…”

“…NAIM is a very elegant technique that allows the simultaneous characterization of chemical groups that are important for RNA function. For comprehensive protocols, we refer to ref [179] and [180]. Here, we just give a brief overview over the general method of NAIM and its applications for the identification of metal ion binding sites in RNA.…”

“…Also, RNAs that have not undergone a selection step should be added as controls to analyze the overall level of analog incorporation into the RNA. If the incorporation level is too high, the bands at the bottom of the gel will be much stronger than those at the top of the gel, as multiple iodine induced cleavages occur in each RNA [180]. Unselected and uncleaved control samples are added to check for background degradation.…”