Detection of aberrant 2'-5' linkages in RNA by anion exchange

Formation of aberrant 2'-5' linkages can unintentionally occur in chemical synthesis of RNA. These linkages may arise by phosphoryl migration during deprotection, release, or subsequent steps during manufacture of therapeutic RNA. Their presence has been linked to a number of biochemical activities, so their potential for contribution to "off-target" effects is significant. Assaying for their presence, to ensure safe and effective therapeutic activity, is not straightforward. Since these linkages do not alter the RNA mass or the ionic or hydrophobic character of the product, confirmation of their presence or absence is not readily addressable by conventional chromatographic, electrophoretic, or mass spectrometric techniques. Since 2'-5' linkages are known to alter RNA solution conformation, they may also alter stationary phase interactions. A method for identifying the presence of these aberrant linkages by pellicular anion-exchange HPLC is presented in this unit.