5. In your opinion, what techniques or methods might become more important in the analysis of oligonucleotides?

Chad Christianson (Alturas Analytics)

The development of sample preparation and chromatographic products from multiple vendors is critical in future oligonucleotide analysis. Competing vendors innovating new sample preparation technologies for plasma and tissue preparation should result in increasing sample throughput and reduced method development time. Instrument vendors will also be important. Sensitive, selective, accurate and precise quantitation of the sample extracts is also required of any MS system analyzing oligonucleotides. Vendors developing next-generation HRMS instruments that have these traits are ideal since they can be more selective due to the high-resolution capability compared to a typical triple quad MS.

 Zamas Lam (QPS)

Hybridization followed by LBA or chromatography for better sensitivity and LC–HRMS for its ability to quantitate and identify metabolites in the same run.

 Ken Cook (Thermo Fisher Scientific)

Bottom-up or top-down sequencing by LC–HRMS, analogous to that which is routinely used for protein therapeutics, will become more and more common as the availability of the methods become known. The nuclease digestions have always been difficult to control but the development of digestion enzymes on magnetic beads allows precise automated control of the start and end of digestion. Direct sequencing of oligonucleotides has never been adopted previously due to the lack of commercial software to automate the annotation of the sequence from HRMS fragmentation data due to the lack of commercial software to automate the annotation of the sequence from HRMS fragmentation data. This is now available and opens up this type of analysis. The quality control [QC] laboratories will be impacted by the need for the extra information possible from the introduction of new easy to use HRMS systems into regulated QC environments.

Alexey Wolfson (Advirna)

Modern oligonucleotide therapeutics are based on heavily modified molecules with a very long half-life in tissues – months to years. For biodistribution studies, development of ELISA-like methods of detection is of great importance. Further development of MS techniques to identify the products of modified oligonucleotide degradation for analysis of their metabolism and long-term toxicity are also important.

The opinions expressed in this feature are those of the authors and do not necessarily reflect the views of Bioanalysis Zone or Future Science Group.