Chemical sequencing of phosphorothioate oligonucleotides using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A new technique for sequencing phosphorothioate oligonucleotides is demonstrated that uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), Current sequencing protocols for phosphorothioate analysis have drawbacks to their widespread implementation. Enzymatic and Maxam-Gilbert sequencing techniques require oxidation to the phosphodiester moiety, thereby increasing analysis time and eliminating the ability to locate modified linkages, Tandem mass spectrometry, which is finding increased use in phosphodiester oligonucleotide sequencing, has not been demonstrated on molecules with phosphorothioate-linked backbones. In the approach presented here, sequencing of the phosphorothioate-linked oligonucleotide is carried out using 2-iodoethanol to cleave at thiolated sites along the backbone, The technique has several advantages over current phosphorothioate sequencing methods: (1) sequencing is performed on the sample without prior oxidation; (2) both 5' --> 3' and 3' --> 5' mass ladders are generated, permitting bidirectional sequencing; (3) it is possible to determine the location of phosphorothioate linkages in mixed phosphorothioate/ phosphodiester oligonucleotides; (4) analysis times are short (less than or equal to 90 s); and (5) small sample amounts are used (<10 nmol). This approach is demonstrated on oligonucleotides with phosphorothioate linkages and should be amenable to the analysis of phosphorodithioates.