Diastereomer\nCharacterization of PS-Modified Synthetic\nOligonucleotides Using Cyclic IMS-MS

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