Playing fast and loose with electrostatic anchors on RNA

Two weeks ago we discussed how to find ligand-binding sites in RNA. Last week we wrote about how difficult it is to find good ligands even for good binding sites in RNA. A recent open-access paper in J. Med. Chem. by Christian Kersten and colleagues at Johannes Gutenberg-University explores why targeting RNA is so tough.

 

The researchers were interested in two well-characterized riboswitches, naturally occurring RNA elements that bind to small molecules such as metabolites. Specifically, they chose to study a riboswitch that binds to S-adenosyl methionine (SAM, structure here) and a riboswitch that binds to prequeuosine-1 (PreQ₁) and prequeuosine-0 (PreQ₀). 

Due to the phosphate backbone, RNA is highly negatively charged. The researchers asked whether positively charged moieties on ligands can serve as “electrostatic anchors” to generally improve affinity, and if so whether this can lead to any design principles. Multiple biophysical techniques were used to study the interactions of the two riboswitches with various natural and synthetic ligands: surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and microscale thermophoresis (MST).

 

In the case of the SAM-VI riboswitch, the researchers compared the binding of SAM with closely related molecules having either one fewer positive charge (S-adenosyl homocysteine, or SAH) or synthetic ligands with the same or one more positive charge than SAM. Not surprisingly, SAM has the highest affinity, binding 20-50 fold more tightly than SAH. Further analysis suggested this is largely driven by an increased association rate, in which the positive charge accelerates the kinetics of binding. The driving energy for binding the ligands is enthalpic, but the favorable electrostatic interactions for more positively charged ligands are largely countered by an entropic penalty.

 

Similarly, the affinity of positively charged PreQ₁ for the PreQ₁ riboswitch is higher than the affinity of neutral PreQ₀, though not dramatically. As in the case of the SAM-VI riboswitch, the association rate of the positively charged ligand is more rapid than that of the neutral ligand. Binding for both ligands is highly enthalpic, with unfavorable entropy.

 

Previous reports had described other synthetic ligands for the PreQ₁ riboswitch, each with between one and three cationic centers. However these ligands showed no binding by ITC, questionable binding by MTC, and non-saturable, non-specific “loose binding” by SPR. Positive charges alone are not sufficient for high affinity, specific binding.

 

So what does it all mean? While adding positive charges can improve affinity of ligands for RNA, the increased affinity is usually not dramatic due to enthalpy-entropy compensation. The researchers note that, even for good ligands, the “thermodynamic binding profiles differ from typical protein-ligand interactions, where enthalpic and entropic contributions are usually more balanced.” Moreover, as we’ve noted, protein ligands often gain significant affinity with entropic gains by displacing "high energy water" molecules, but such opportunities are likely less common on the polar surface of RNA.

 

The affinity and ligand efficiency of PreQ₁ for its riboswitch are impressive, so clearly it is possible for small drug-like ligands to bind tightly to RNA. But this interaction is the product of countless eons of evolution. This careful paper suggests why building similarly effective synthetic ligands for most RNA will be difficult.