Identification of RNA linkage isomers by anion exchange purification with electrospray ionization mass spectrometry of automatically desalted phosphodiesterase-II digests

Among the possible contaminants unique to RNA are linkage isomers that are difficult to identify by standard oligonucleotide analysis techniques. In a prior study, we used nonporous and monolithic polymer anion exchangers for purification and demonstrated a method to identify the presence of the linkage isomers. We also suggested a confirming technique employing phosphodiesterase-II (PDase-II), an enzyme incapable of cleaving 2′–5′ linkages. We now present a method identifying the location of the linkage in the RNA isomer by anion exchange purification and electrospray ionization mass spectrometry (ESI–MS) of the digestion products. Because the ion-pair reversed-phase liquid chromatography (IP–RPLC) desalting methods we previously employed do not effectively separate oligonucleotides less than 6 bases from salt, we employed a direct reversed-phase method to automatically desalt the digestion products and then assessed the desalted digests by ESI–MS. The length and base composition of the fragments identified indicate that PDase-II cleaves up to and skips over the aberrant linkage and then resumes cleavage 1 or 2 bases to the 3′ side of the 2′–5′ linkage.