The use of strong anion-exchange (SAX) magnetic particles for the extraction of therapeutic siRNA and their analysis by liquid chromatography/mass spectrometry.

Traditional methods for extracting oligonucleotides from serum and other biological fluids are often time-consuming and require multiple steps. Magnetic particle based separation of oligonucleotides has gained importance recently due to the advantages of simplicity and high efficiency. Here we report the development and optimization of commercially available strong anion-exchange (SAX) magnetic beads for the extraction of siRNA from human serum. The beads allowed for rapid extraction of siRNA from human serum in 100-200 μL of liquid chromatography/mass spectrometry (LC/MS)-compatible buffer in less than 1 h for a 96-well plate with no further drying steps. Due to the strong cation-binding properties of oligonucleotides, volatile ammonium salts such as triethylammonium bicarbonate (TEAB), ammonium bicarbonate, and NH(4) Cl were used to elute the siRNA from the beads. For more hydrophobic siRNA sequences, the addition of 5-10% organic solvent was required for elution. The recovery of chemically modified siRNA from human serum was around 80% for two types of beads examined; however, the recovery for highly modified sequences differed greatly between the two types of beads. In addition to extracting highly modified oligonucleotides, the SAX beads were also able to extract liposomal formulated siRNAs from serum with no interference from the lipid formulation. The extraction of siRNA from human serum was linear over the tested range of 50 ng/mL to 10 µg/mL. Using this extraction methodology, we have created a workflow to monitor siRNA serum stability by LC/MS. Initial observations confirm that RNase A type degradation with strand cleavage on the 3' side of uridine or cytosine is the dominant cleavage pattern in serum. This finding has implications for the selection and modification of therapeutic siRNAs and demonstrates the utility of magnetic beads as a simple and rapid extraction technique for siRNA.