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Bioanalysis. 2011 Mar;3(5):499-508. doi: 10.4155/bio.11.18.

Dual ligation hybridization assay for the specific determination of oligonucleotide therapeutics.

Author information

  • 1Charles River Preclinical & Clincal Services Montréal Inc., 22022 Transcanadienne, Senneville, QC H9X3R3, Canada. guy.tremblay@crl.com

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.

PMID:
21388263
[PubMed - indexed for MEDLINE]
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