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

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Therapeutics

Background:

• Ribozymes, or ribonucleic acids with enzymatic functions, have limited practical applications in nucleic acid medicine due to low activity.
• Mimicking the catalytic mechanisms of ribonuclease A is a potential strategy to enhance functional RNA activity.

Purpose of the Study:

• To obtain histidine-dependent functional RNAs with high activity by employing in vitro selection with L-histidine.
• To investigate the catalytic mechanisms and structural roles of histidine in the selected RNA molecules.

Main Methods:

• Construction and in vitro selection of a 70-nucleotide random sequence RNA library using L-histidine.
• Characterization of self-cleaving RNA variants, including kinetic analysis (rate constants) and assessment of histidine specificity.
• pH profile analysis to elucidate the role of histidine in catalysis and structure.

Main Results:

• A histidine-specific self-cleaving RNA (I-04) with a rate constant of 4.6 × 10⁻³ min⁻¹ at pH 5.0 was isolated.
• Further selection yielded II-28, exhibiting histidine-dependent cleavage activity (rate constant 3.0 × 10⁻³ min⁻¹ at pH 5.0) but no activity with histidine analogs.
• The obtained ribozymes demonstrated low activity, distinct from ribonuclease A, and were independent of divalent metal ions. Histidine's role was presumed to be structural rather than catalytic.

Conclusions:

• Histidine-dependent, metal-ion-independent self-cleaving RNAs were successfully selected.
• The selected ribozymes exhibit low catalytic activity, and histidine appears to stabilize the active structure rather than acting as a catalytic residue.
• Further research is needed to enhance ribozyme activity for practical applications in nucleic acid medicine.