CHEMICAL SEQUENCING OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING MATRIX-ASSISTED LASER-DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY

A new technique for sequencing phosphorothioate oligonucleotides is de monstrated that uses matrix-assisted laser desorption/ionization time- of-flight mass spectrometry (MALDI-TOFMS), Current sequencing protocol s for phosphorothioate analysis have drawbacks to their widespread imp lementation. Enzymatic and Maxam-Gilbert sequencing techniques require oxidation to the phosphodiester moiety, thereby increasing analysis t ime and eliminating the ability to locate modified linkages, Tandem ma ss spectrometry, which is finding increased use in phosphodiester olig onucleotide sequencing, has not been demonstrated on molecules with ph osphorothioate-linked backbones. In the approach presented here, seque ncing of the phosphorothioate-linked oligonucleotide is carried out us ing 2-iodoethanol to cleave at thiolated sites along the backbone, The technique has several advantages over current phosphorothioate sequen cing 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 th an or equal to 90 s); and (5) small sample amounts are used (<10 nmol) . This approach is demonstrated on oligonucleotides with phosphorothio ate linkages and should be amenable to the analysis of phosphorodithio ates.