Thermodynamics and kinetics of RNA tertiary structure formation in the junctionless hairpin ribozyme

- SPR studies of domain docking kinetics and thermodynamics in the hairpin ribozyme
- The reaction is strongly enthalpy-driven in the native ribozyme.
- High activation barriers are consistent with structural rearrangements.
- O-methylation at the cleavage site significantly alters the reaction energetics.
- RNA tertiary structure formation displays a diversity of thermodynamic signatures.

The hairpin ribozyme consists of two RNA internal loops that interact to form the catalytically active structure. This docking transition is a rare example of intermolecular formation of RNA tertiary structure without coupling to helix annealing. We have used temperature-dependent surface plasmon resonance (SPR) to characterize the thermodynamics and kinetics of RNA tertiary structure formation for the junctionless form of the ribozyme, in which loops A and B reside on separate molecules. We find docking to be strongly enthalpy-driven and to be accompanied by substantial activation barriers for association and dissociation, consistent with the structural reorganization of both internal loops upon complex formation. Comparisons with the parallel analysis of a ribozyme variant carrying a 2′-O-methyl modification at the self-cleavage site and with published data in other systems reveal a surprising diversity of thermodynamic signatures, emphasizing the delicate balance of contributions to the free energy of formation of RNA tertiary structure.

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