Oligonucleotide Sequencing by Mass Spectrometry with New OligoSeq Ion Calculator

by Dr. Robert Duff, Manager of the Biochemistry Department

Demands for purified synthetic oligonucleotides have increased due to their use in applications such as primers in polymerase chain reactions, as artificial genes for sitedirected mutagenesis studies, as probes for in-situ DNA hybridization and also potential therapeutic use as antisense DNA/RNA strands, aptamers and most notably, RNAi. Clinical use of these molecules requires a high degree of quality control, including sequence confirmation and the identification of minor impurities/degradants. Additionally due to the nuclease resistant composition of certain oligonucleotides, older techniques of sequencing as based on nuclease degradation are precluded. Newer analytical techniques that use mass spectrometry must be developed for the pre-clinical evaluation of nucleic acid-based therapeutic products.

Each facility or pharmaceutical company will be required to test if their synthesized oligonucleotide is truly what they claim it to be. It is critical that the synthesized sequence be confirmed as not to contain base reversals or chemical mutations that if left unnoticed could have a devastating effect on the bioefficacy of the oligonucleotide and the therapeutic outcome of a clinical trial. The software used to interpret the raw data from the sequencing by mass spectrometry is definitely behind the available peptide sequencing software.

To bridge this gap and aid in this process, a proprietary program was written and built within the Laboratory Information Management System at Lancaster Labs. The program is named OligoSeq Ion Calculator, and it is able to calculate the accurate mass and CID fragments of modified or unmodified oligonucleotides. There are no restrictions for the number or type of modifications on the oligonucleotide. Furthermore, unlike on-line calculators, there is no upper size limit to the oligonucleotide used in this calculation. Overall, this program aids the analyst in the sequencing of the oligonucleotide by predicting the mass-to-charge for the many characteristic “a,” “a- B,” “b,” “d,” “w” and “y” ions formed by collision induced dissociation. A table is produced that contains the theoretical CID fragments. The sequence is then matched to the fragments for 100 percent coverage. Clearly the need for sequence confirmation exists and with the appropriate method and software, this testing can be made routine and validated.