L-Histidine Modulates the Catalytic Activity and Conformational Changes of the HD3 Deoxyribozyme.

Abstract

Background/Objectives: Riboswitches are functional nucleic acids that regulate biological processes by interacting with small molecules, such as metabolites, influencing gene expression. Artificial functional nucleic acids, including deoxyribozymes, have been developed through in vitro selection for various catalytic functions. In a previous study, an l-histidine-dependent deoxyribozyme was identified, exhibiting RNA cleavage activity in the presence of l-histidine resembling ribonuclease catalytic mechanisms. This study aims to clarify the role of l-histidine in the activity and structural formation of the l-histidine-dependent deoxyribozyme (HD), focusing on the binding properties and conformational changes of its derivative HD3. Methods: Conformational changes in HD3 were analyzed using circular dichroism (CD) under varying concentrations of l-histidine. Direct binding analysis was conducted using carbon-14 (¹⁴C)-labeled l-histidine and a liquid scintillation counter. The catalytic activity of HD3 in the presence of different l-histidine concentrations was measured. Results: The binding constant for l-histidine-induced conformational changes (K_a(CD)) was found to be 2.0 × 10³ (M^-1), whereas for catalytic activity (K_a(Rxn)) and scintillation counting (K_a(RI)), it was approximately 1.0 × 10³ (M^-1). Conclusions: l-Histidine plays an essential role in both the catalytic activity and structural formation of the HD3 deoxyribozyme. The consistent binding constants across different experimental methods highlight the significant contribution of l-histidine to the active folding of deoxyribozymes.