Trinucleotide repeat system for sequence specificity analysis of RNA structure probing reagents.

Chemical and enzymatic structural probes have been used for decades to obtain rapid and comprehensive information regarding the molecular architecture of various RNAs. Despite their widespread use, the sequence specificity of these RNA structural probing reagents has not yet been thoroughly characterized. In this study, we revisited the properties of commonly used structural probes such as Pb(II) ions, ribonuclease V1, ribonuclease T2, and the S1 and mung bean nucleases by testing them on highly regular triplet repeat sequences representing phosphodiester bonds with every possible combination of 3' and 5' adjacent nucleotides. We show that Pb(II) ions preferentially cleave after pyrimidines and that S1 nuclease possesses a previously overlooked specificity toward phosphodiester bonds following G residues. We also observed that mung bean nuclease shows a preference for cleaving ApN bonds and that RNase V1 mainly recognizes U residues in both single- and double-stranded RNAs. These data are important for accurate interpretation of the results of structure probing experiments and for assignment of the correct structure to individual RNA molecules. The triplet repeat transcript system described here may be considered as a reliable platform for determining the sequence specificity of other reagents used to probe RNA structure.