Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site A

Area of Science:

• Biochemistry
• Computational Biology
• Molecular Biophysics

Background:

• Predicting RNA-ligand interactions is crucial for drug discovery.
• Small-molecule ligands often target specific RNA structures.
• Computational methods combined with experimental assays offer a powerful approach.

Purpose of the Study:

• To validate a computational methodology for predicting minimum RNA structural requirements for ligand binding.
• To apply this methodology to the aminoglycoside antibiotic paromomycin.
• To determine the minimum RNA duplex size for high-affinity paromomycin binding.

Main Methods:

• Molecular dynamics (MD) simulations.
• Free-energy calculations.
• In vitro binding assays (EC50 determination).
• Simulations of an 11-mer:10-mer duplex containing a 16S ribosomal RNA decoding A-site bound to paromomycin.

Main Results:

• The computational methodology predicted high-affinity binding of paromomycin to the 11-mer:10-mer RNA duplex.
• Smaller RNA duplexes were predicted to lack complex stability and binding affinity.
• Experimental validation confirmed high-affinity binding (EC50 = 0.28 microM) for the 11-mer:10-mer duplex and no binding for smaller complexes.
• MD simulations accurately reproduced experimental dynamic and structural properties of the RNA A-site.

Conclusions:

• The validated computational methodology can predict minimum RNA structural motifs for ligand binding.
• MD simulations are a valuable supplement to in vitro methods for RNA-drug interaction studies.
• This approach is applicable to both small, rigid ligands and large, flexible ligands like paromomycin.