Functional Analysis of Histidine-Dependent Self-Cleaving RNAs Obtained by In Vitro Selection

Ribozymes, ribonucleic acids with enzymatic functions, have not yet been practically used in nucleic acid medicine, possibly due to their low activity levels. We hypothesized that functional RNAs with high activity can be obtained by mimicking the catalytic reactions of ribonuclease A. We aimed to obtain histidine-dependent functional RNAs from an RNA library using in vitro selection with L-histidine. An RNA library with a random sequence of 70 nucleotides was constructed and used. A self-cleaving RNA (I-04) with histidine specificity and a rate constant of k_obs(I-04) = 4.6 × 10^-3 (min^-1) at pH 5.0 was obtained. Owing to its low activity, reselection was performed. II-28, obtained on the second selection, showed cleavage activity in the presence of histidine, but not with histidine analogs. The rate constant of II-28 was k_obs(II-28) = 3.0 × 10^-3 (min^-1) at pH 5.0, comparable to that of I-04. The activity of these molecules, however, is very low and different from that of ribonuclease A. Several histidine-dependent and divalent metal ion-independent self-cleaving RNAs were obtained. The pH profile of the RNAs suggested that histidine was not utilized as a catalytic residue, but histidine was presumed to be working in the formation of the active structure.