There are currently two main approaches to extracting oligonucleotides from biological matrices: liquid/liquid extractions using Phenol:Chloroform:Isoamyl Alcohol (25:25:1) and SPE. Both approaches use the anionic nature of the phosphorous group that bridge the series of nucleobases, producing extracts with a nonspecific mixture of acidic compounds. Both approaches have proven to be successful in high throughput environments, but they can be labor intensive due to the multiple steps involved which may lead to low recoveries and long days in the lab. An alternate approach is suggested here that instead of focusing on the charge of the oligonucleotide, it may be preferable to focus on the specific base pairs that comprise the oligonucleotide through a hybridization approach that has already proven to be successful in LBA based assays. This would allow a common workflow that would enable a direct capture of the oligonucleotide of interest and reduce potential interference from the sense strand in siRNA therapies. The approach would also be applicable to the full class of oligonucleotides such as siRNA, ASOs, PPMOs, and PNAs.
Learning Objectives:
Upon completion, participant will be able to understand the major obstacles related to the bioanalysis of oligonucleotides.
Upon completion, participant will know the two basic approaches for extracting oligonucleotids from a biological matrix.
Upon completion, participant will understand a new approach to extracting oligonucleotides from a biological matrix using hybridization followed by LC/MS analysis and the potential benefit of this highly selective approach.