Dual ligation hybridization assay for the specific determination of oligonucleotide therapeutics

Background: Oligonucleotide-based therapeutics are quantified with hybridization assays in biological matrices such as plasma and tissues. Current hybridization methods do not entirely discriminate the parent compound from 5´- or 3´-N-X truncated metabolites. Results: A dual ligation-based hybridization assay was developed to circumvent the limitations of current assay formats. Ligation of probes at either end of the analyte is performed via a bi-enzymatic reaction consisting of polynucleotide kinase and DNA ligase. The method was validated with regard to mechanism, specificity, precision and accuracy. Conclusion: The dual ligation assay is specific for the parent compound and detects the full-length product with intact...