Fragments vs RNA with SPR: A guide

Fragment-based lead discovery on RNA has a long history: the first mention on Practical Fragments was in 2009. Most often, various NMR methods have been used (see this example from last year), though isothermal titration calorimetry (ITC) is also effective. However, both of these techniques generally require considerable amounts of RNA. In a recent Biochemistry paper, J. Winston Arney and Kevin Weeks describe using SPR, which could increase the speed and ease of screening RNA.

 

Non-specific binding is a significant problem in characterizing RNA ligands. RNA is negatively charged, and many ligands are positively charged, leading to non-specific interactions. In a typical SPR experiment, the target is bound to a surface and the analyte is allowed to flow over the immobilized target; binding causes a change in refractive index that can be detected. However, if the analyte interacts non-specifically with the target, this will also be detected. For high affinity ligands the non-specific interactions may be minimal at low concentrations, but for low-affinity ligands such as fragments, it can be difficult to differentiate specific from non-specific binding.

 

SPR experiments generally use a reference cell, in which the analyte is allowed to flow over the surface in the absence of target; this signal is then subtracted from the target channel. Arney and Weeks decided to use a reference cell containing mutant RNA not expected to bind to the ligand.

 

The researchers developed their approach using two different riboswitches, each with known high-nanomolar ligands. Immobilizing the riboswitches to the chip and flowing ligand led to non-specific binding at concentrations of 100 µM or so. However, when the reference cell contained a mutant riboswitch designed not to bind to the ligands, this non-specific binding could easily be subtracted, leading to simple single-site binding models.

 

Of course, creating a mutant RNA assumes you already know where your ligand binds, which is not true if you are looking for ligands to a new target. To increase the generality of their approach, the researchers used a different riboswitch or a completely arbitrary RNA for the reference. These also worked, though not quite as well as the targeted mutants.

 

Finally, the researchers tested a dozen RNA-ligand pairs that had previously been rigorously characterized. Importantly, these varied considerably in affinity, from 8 nM to 2 mM. Most of them were also fragment-sized, with molecular weights as low as 119 Da. The correlation between SPR dissociation constants and those reported in the literature was excellent.

 

The technique does have limitations. First, the RNA-bound surfaces do seem somewhat unstable over a period of days. Also, larger RNAs present technical challenges, though the researchers do state that they have been able to examine molecules as large as 300 nucleotides. Overall this looks like a nice approach for measuring RNA-ligand affinities.