Fast Binding Kinetics of RNA Aptamers Measured using a Novel Microfluidic Mixer

Abstract

RNA aptamers that target specific biomolecules with high specificity are growing in popularity due to their ease of production compared to antibodies and their tight binding. Aptamers have uses ranging from targeted protein inhibition to the creation of new fluorescent labels. One example of the latter is the Spinach aptamer which binds 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) to form a fluorescent complex resembling the chromophore of GFP. Upon binding its target, the RNA aptamercauses a conformational stabilization of DFHBI resulting in a fluorescent complex. The kinetics of Spinach aptamer binding and activation of the fluorophore have not been thoroughly investigated. Using a novel microfluidic fast mixing device which has been shown to have dead time for mixing of ∼500 μs and be especially useful for viscous solutions, we have examined the on-rate of the Spinach/DFHBI binding reaction to gain an increased understanding the kinetics of aptamer-ligand binding.