MONITORING YEAST tRNA ADSORPTION BY QCM-D: AN OPPORTUNITY FOR OPTIMIZATION OF APTAMER SELECTION CONDITIONS

Abstract

RNA aptamers that bind to a wide range of targets with high affinity and specificity have been identified via the in vitro systematic evolution of ligands by exponential enrichment (SELEX). However, the process is quite unpredictable due in part to binding that occurs not only on the targets themselves but also on any of the other functional groups, moieties, or surfaces. Recent modelling work has shown that this level of “background binding” is a key parameter in the performance of aptamer selection processes. One strategy to minimize the amount of background binding is to pre-block those possible binding sites with a non-amplifiable nucleic acid molecule, such as yeast tRNA. It is also known that binding buffer conditions have strong effect on the binding affinity of nucleic acids. However, there are no detailed studies and little quantitative information available to guide the design of aptamer selection processes. In this study, the binding ability of yeast tRNA, which has comparable size with most RNA aptamer libraries, on both silicon dioxide and poly (ethylene terephthalate glycol) (PET-G) surfaces was studied using Quartz Crystal Microbalance with Dissipation (QCM-D). Silicon dioxide surface is a commonly used substrate for QCM-D tests on the adsorption behaviour of different nucleic acid. PET-G is a commonly used polymer substrate for the fabrication of microfluidic devices, which are advanced techniques for aptamer selection. The presence of specific divalent cations, for example Mg2+ over Ca2+, in binding buffers greatly enhanced the binding of yeast tRNA on silicon dioxide surfaces and PET-G surfaces. Proper NaCl concentration (100 mM) and MgCl2 concentration (5 mM) is necessary to enhance yeast tRNA binding on both surfaces. Yeast tRNA binding ability on silicon dioxide surfaces show more dependence on binding buffer pH than on PET-G surfaces.