Exposing Hidden High-Affinity RNA Conformational States.

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• RNA binding affinity is influenced by conformational changes and associated energy costs, termed "conformational penalties."
• Measuring these penalties is challenging as bound RNA conformations often have undetectable populations in the unbound state.

Purpose of the Study:

• To develop a thermodynamic model for coupled RNA conformational change and ligand binding.
• To quantify conformational penalties in the HIV-1 TAR RNA-argininamide (ARG) interaction model system.

Main Methods:

• Utilized Nuclear Magnetic Resonance (NMR) chemical shift perturbations and relaxation dispersion.
• Applied Bayesian inference to a 12-state model to estimate energies of low-population states.
• Investigated the interaction between HIV-1 TAR RNA and argininamide (ARG) as a Tat protein mimic.

Main Results:

• Identified a minimum of four distinct RNA intermediates based on TAR conformation and ARG occupancy.
• Revealed that the dominant bound TAR conformation, with two ARG ligands, has a below-detection limit equilibrium population unbound.
• Demonstrated that ARG binds the pre-folded TAR conformation with nanomolar affinity, despite a weak apparent overall affinity (0.2 mM).

Conclusions:

• Conformational penalties significantly determine RNA-ligand binding affinity and can drive binding cooperativity.
• Accurate prediction of RNA recognition requires considering the energy costs of conformational transitions.
• Findings have implications for RNA-targeted drug discovery and understanding RNA-protein interactions.