Origins of Reactivity in SAM-Utilizing Ribozyme SAMURI-Catalyzed RNA Alkylation

Area of Science:

• Biochemistry and Molecular Biology
• RNA Catalysis and Engineering
• Chemical Biology

Background:

• Programmable RNA catalysts are crucial for advancing RNA functionality and therapeutics.
• The SAM analogue-utilizing ribozyme (SAMURI) facilitates site-specific RNA alkylation using S-adenosylmethionine (SAM) or synthetic propargylic Se-2,6-diaminopurinribosyl-selenomethionineamide (ProSeDMA).
• The precise molecular mechanisms governing SAMURI's reactivity are not fully understood.

Purpose of the Study:

• To elucidate the conformational and electronic factors dictating SAMURI's catalytic efficiency.
• To provide a mechanistic framework for designing improved programmable RNA alkyltransferases.

Main Methods:

• Integrated computational approaches including molecular dynamics, 3D-RISM solvation analysis, alchemical free energy calculations, and ab initio QM/MM free energy simulations.
• Analysis of conformational dynamics, active site interactions, and electronic properties of the cofactor and substrate.
• Prediction of quantum pKa shifts to assess the impact of atomic substitutions on nucleophilicity.

Main Results:

• SAMURI maintains a stable global fold, with catalysis dependent on accessing rare, catalytically competent near-attack configurations (freact).
• A Mg2+ binding site and a specific hydrogen bond involving the cofactor's amine group are identified as key factors enriching freact.
• ProSeDMA exhibits higher reactivity than SAM due to superior leaving group properties, and atomic modifications at A52 enhance nucleophilicity and catalytic rate (kint).

Conclusions:

• SAMURI catalysis is a result of both conformational preorganization and electronic effects.
• The findings offer a foundation for designing novel RNA alkyltransferases with enhanced catalytic properties.
• Understanding these design principles is critical for expanding the therapeutic and functional applications of RNA technology.