Characterization of novel cofactorless self-cleaving RNAs with activity at low pH

Several diseases abnormally lower cellular pH owing to metabolic pathway changes. Developing drugs based on genetic material that act only in target tissues may reduce side effects by exploiting the differences between diseased and normal tissue environments. Most naturally occurring ribozymes-ribonucleic acids with catalytic activity-are active at a neutral pH and inactive at lower pH. Ribozymes that cleave RNA only in acidic solutions require a general acid with a sufficiently low pK _a and a general base with a pK _a closer to the acidic region. As the rational design of such a ribozyme is difficult, we performed an in vitro assay for the selection of self-cleaving RNAs under acidic conditions. We attempted to develop a self-cleaving RNA that only acts in acidic solutions without requiring divalent metal cations, as the activity of natural ribozymes is affected by the presence or absence of these ions. To obtain RNA molecules with high activity under any conditions, in vitro selection was performed in the absence of divalent metal ions and cofactors. Using this method, we obtained a new functional RNA with self-cleavage activity only at an acidic pH. This RNA also exhibited high activity in the absence of divalent metal ions. This study derived a novel functional RNA. The obtained RNAs were shortened to a 53-nucleotide RNA with the original properties, and the sequence required for the catalytic activity was identified.