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 kobs(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 kobs(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.

Tissue-resident memory T cells (TRM) remain in nonlymphatic barrier tissues for extended periods and are deeply involved in immune memory at the site of inflammation. Here, we employed multilayered single-cell analytic approaches including chromatin, gene, and protein profiling to characterize a unique CD4+ TRM subset present in the inflamed gut mucosa of Crohn's disease patients. We identified two key transcription factors, RUNX2 and BHLHE40, as regulators of pathologically relevant CD4+ TRM. These transcriptional regulators work together to induce distinct cellular properties of disease-specific TRM, such as cytotoxicity, T helper 1-effector activity, and tissue retention. Downregulation of RUNX2 and BHLHE40 in patient-derived gut CD4+ T cells resulted in the mitigation of the pathogenic phenotype of these cells. Conversely, the ectopic overexpression of both transcription factors in healthy donor-derived CD4+ T cells drove IFN-γ pathways and enhanced tissue residency. Our findings illuminate the transcriptional programs driving disease-specific T cell formation in Crohn's disease.