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

Area of Science:

• Pharmacokinetics and Drug Development
• Bioanalytical Chemistry
• Molecular Diagnostics

Background:

• Quantitative bioanalysis is crucial for pharmacokinetic studies during drug development.
• Conventional methods for analyzing antisense oligonucleotides (ASOs) face challenges in sensitivity, specificity, and complexity.
• There is a need for improved analytical techniques to accurately quantify ASOs and their metabolites.

Purpose of the Study:

• To evaluate a novel nonenzymatic hybridization assay utilizing probe alteration-linked self-assembly reaction (PALSAR) technology for ASO quantification.
• To assess the sensitivity, specificity, accuracy, and precision of the PALSAR method for ASO detection in biological matrices.
• To demonstrate the capability of PALSAR in distinguishing ASO metabolites with high specificity.

Main Methods:

• Development and application of a probe alteration-linked self-assembly reaction (PALSAR) assay.
• Nonenzymatic hybridization assay utilizing PALSAR as a signal amplification strategy.
• Quantification of ASOs in mouse tissue and plasma samples.

Main Results:

• PALSAR achieved high sensitivity for ASO quantification, with a range of 1.5 to 6 pg/ml.
• The assay demonstrated excellent intra-/interday accuracies (86.8-119.1% and 88.1-113.1%, respectively) and precision (≤17.2%).
• Crossreactivity with a single base difference metabolite (3'n-1) was less than 1%, indicating high specificity.

Conclusions:

• The PALSAR approach provides a sensitive and specific method for detecting antisense oligonucleotides (ASOs).
• This technology offers an effective solution for overcoming challenges in conventional ASO bioanalysis.
• PALSAR is a promising tool for distinguishing metabolites and advancing drug development studies.