An automated algorithm for sequence confirmation of chemically modified oligonucleotides by tandem mass spectrometry.
We have developed a tandem mass spectrometry (MS/MS) data analysis program for confirmation of sequence of chemically modified oligonucleotides. The method is based on the analysis of deconvoluted MS/MS data for fragment ions from three charge states and comparison of these data against a set of computer-generated masses from expected fragmentation patterns. The algorithm compares the experimental masses not only against the fragment set predicted for the expected sequence but also against a wider test set covering all next-neighbor position switches of the original sequence and all pairwise swaps of nucleosides, which in synthesis would result in molecules with masses within a preset mass tolerance. The algorithm is capable of identifying incorrect sequences that would not be distinguished by identity testing with electrospray ionization mass spectrometry. The method has been tested with permutations of the two 21-mer single strands of a chemically modified short interfering RNA containing 2'-O-methyl and phosphorothioate linkages. For both strands, challenge sequences were synthesized and tested with the premise that they were the original sequences. The algorithm correctly reported the locations of next-neighbor position switches and nucleoside swaps. The results confirm the approach as useful for MS/MS-based identity test methods for synthetic oligonucleotides.
Kretschmer
Matthias
M
Alnylam Pharmaceuticals, Cambridge, MA 02142, USA. mkretschmer@alnylam.com
Lavine
Gary
G
McArdle
James
J
Kuchimanchi
Satya
S
Murugaiah
Veeravagu
V
Manoharan
Muthiah
M
D000465:Algorithms; D001483:Base Sequence; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
D009841:Oligonucleotides