Synthesis And Testing Of Novel Boronic Acid Columns For The Isolation Of Micro-RNA Molecules And Their Associated Binding Proteins

Recently, it has been found that in mammals circulating microRNA’s can be used as very effective, early predictors of a variety of disease states.  The function of these circulating microRNAs is currently unknown.  Our laboratory has examined microRNAs in human, mouse, rat, and horse by RNA extraction from serum or plasma.  Methods normally required for RNA extraction are very harsh and do not allow the study of functional microRNA complexes.  Thus the goal of my project is to develop a method by which functional microRNA complexes may be recovered from cell culture media, serum, plasma and other biological fluid.  We hypothesize that an affinity column based on a novel use of boronate will provide a new method to isolate functional microRNA complexes.  This will be accomplished by a synthesis protocol allowing for a novel boronate and polyamine resin. Once synthesized the boronate-polyamine beads will be placed in a column which will allow us to improve  the collection of circulating microRNAs.  In order to characterize the recovered microRNA complexes, we use quantitative RT-PCR and western analysis.  Quantitative RT-PCR determined how effective the column is at isolating microRNAs and western analysis allows the detection of proteins known to be associated with circulating microRNAs.   In order to determine if functional complexes are obtained we treat cells growing in culture and determine if microRNA targets such as p27/CDKN1B expression is altered.  We expect that in the future this type of boronate affinity column may be useful for detecting predictors of disease in patient samples.