Sensitive and specific quantification of antisense oligonucleotides using probe alteration-linked self-assembly reaction technology

Abstract

Quantitative bioanalysis is essential when establishing pharmacokinetic properties during the drug development process. To overcome challenges of sensitivity, specificity and process complexity associated with the conventional analysis of antisense oligonucleotides (ASOs), a new approach to nonenzymatic hybridization assays using probe alteration-linked self-assembly reaction (PALSAR) technology as a signal amplifier was evaluated. PALSAR quantification of ASOs in mouse tissue and plasma was able to achieve a high sensitivity ranging from 1.5 to 6 pg/ml, intra-/interday accuracies in the range of 86.8–119.1% and 88.1–113.1%, respectively, and precision of ≤17.2%. Furthermore, crossreactivity of 3′n-1, a metabolite with a single base difference, was <1%. Our approach provides an auspicious method for distinguishing metabolites and detecting ASOs with high sensitivity and specificity.

Keywords

• antisense oligonucleotide
• bioanalysis
• ligand-binding assay
• nonenzymatic hybridization assay
• PALSAR technology
• sensitivity