Determination of the refractive index increments of small molecules for correction of surface plasmon resonance data

The refractive index increments (RIIs) of several important low-molecular-weight compounds that bind to DNA or RNA were determined with a differential refractometer for correction of data obtained on surface plasmon resonance (SPR) biosensors. Although the ability to investigate small molecule-macromolecule interactions by SPR is relatively new, the technique is rapidly becoming a primary method to screen focused combinatorial libraries and to quantitatively characterize the interactions between compounds identified as binders and target macromolecules. The most widely used SPR analysis software, BIAevaluation (Biacore, Inc.), assumes that the RIIs of ligand and macromolecule are identical. While the assumption is reasonable for studies involving like molecules such as protein-protein interactions, results presented here demonstrate that RII values for small molecules can be significantly different than those of protein or nucleic acid receptors. The results also show that the RII values can vary greatly depending on the structure of the small molecule. Indeed, the RIIs of the molecules investigated here differ by a factor of 2. Any difference in the RII of interacting molecules must be considered for complete analysis of SPR data. Failure to correct for RII differences can result in serious error in data interpretation, especially for systems involving a ligand:receptor stoichiometry greater than 1. The results serve as the beginning of an SPR correction database for the RIIs of small molecules. Additionally, the results can be used to approximate the RIIs of a variety of other small molecules.