Dual ligation hybridization assay for the specific determination of oligonucleotide therapeutics

  • Bioanalysis. 2011 Mar;3(5):499-508. doi: 10.4155/bio.11.18.
Ga Tremblay  1 ,  G Khalafaghian ,  J Legault ,  P Nielsen ,  Aj Bartlett
Affiliations
  • 1. Charles River Preclinical & Clincal Services Montréal Inc., 22022 Transcanadienne, Senneville, QC H9X3R3, Canada. [email protected]
PMID: 21388263 DOI: 10.4155/bio.11.18
Abstract

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 5´- and 3´-ends. The dual ligation assay can also be used to specifically determine individual metabolites in complex mixtures and is currently implemented to quantitative PCR.

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