Diastereomer Characterization of PS-Modified Synthetic Oligonucleotides Using Cyclic IMS-MS

Synthetic oligonucleotides have emerged as effective therapeutics that regulate gene expression to treat and prevent diseases. Oligonucleotide therapeutics are often modified with a substitution of a phosphorothioate (PS) linkage along the phosphodiester backbone to improve the drug performance and stability. The PS modification creates a mixture of diastereomer structures, increasing by a factor of 2<i>ⁿ</i> where <i>n</i> is the number of PS linkages. Despite recent draft guidances highlighting the importance of their characterization, analytical methods to measure the resulting diastereomers are currently lacking. Here, we present a method combining tandem mass spectrometry (MS) and tandem ion mobility spectrometry (IMS) using a cyclic IMS-MS instrument to study diastereomers in PS-modified oligonucleotides. This approach requires no enzymatic digestion as the intact oligonucleotides are directly injected into the MS instrument. Analogous to top-down proteomics, MS fragmentation of the intact oligonucleotide results in 3' and 5' fragment ends that have fewer diastereomers than their intact counterpart. Tandem IMS allows for mobility resolution of the diastereomers at the terminal ends. We tested four model oligonucleotides that differ in either the number of PS bonds or sequence to demonstrate the capability of this method to elucidate diastereomer structures on modified oligonucleotides.