Phenyl boronate column fractionation of high molecular weight RNA

Abstract

The interactions of borate ion with 1,2-cis-diol groups are useful in fractionating mixtures of biological molecules. Separation of RNAs, up to the size of tRNAs, from other biomolecules using phenyl boronate agarose (PBA) column has been shown. In this work, we used phenyl-boronate agarose column to separate normal E. coli 16S ribosomal RNA from 16S ribosomal RNA with a blocked 3'-terminus. The blocking group was a 3',5'-cytidine (5'-32p)-bisphosphate. Using a 30 um boronate/ml of agarose (PBA-30) column we obtained reproducible separations at 22C. The results show that this method is useful in preparing high molecular weight RNA of high specific activity for sequence analysis. It is also effective for separation of nuclease digestion products.