RNA-Ligand Interactions Quantified by Surface Plasmon Resonance with Reference Subtraction.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Structured RNAs are crucial drug targets due to their ligand-binding capabilities.
• Existing analytical methods for RNA-ligand interactions often require substantial material and struggle with complex RNAs.
• Standard Surface Plasmon Resonance (SPR) experiments are hindered by non-specific electrostatic interactions, complicating the analysis of weak RNA binders.

Purpose of the Study:

• To develop a refined SPR method for accurate quantification of specific RNA-ligand binding affinities.
• To overcome limitations of standard SPR by addressing non-specific binding.
• To enable efficient screening of small-molecule ligands for RNA targets.

Main Methods:

• Implementation of SPR with a reference channel containing a non-binding control RNA.
• Subtraction of non-specific binding contributions using the control channel data.
• Validation using small-molecule binders for two riboswitch RNAs with a range of affinities (nanomolar to millimolar).

Main Results:

• The reference subtraction method accurately measures specific RNA-ligand binding affinities.
• This approach effectively distinguishes specific binding from non-specific electrostatic interactions.
• The method demonstrated reliability for low-molecular-mass fragment ligands and weak binders.

Conclusions:

• SPR with reference subtraction provides a robust and efficient method for characterizing RNA-ligand interactions.
• This technique conserves valuable RNA and ligand material.
• It facilitates rapid exploration of the ligand-binding landscape for diverse RNA targets, advancing drug discovery efforts.