Dual ligation hybridization assay for the specific determination of oligonucleotide therapeutics

Bioanalysis. 2011 Mar;3(5):499-508. 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.

MeSH terms

  • Base Sequence
  • Calibration
  • DNA Ligases / metabolism
  • Humans
  • Immunoassay
  • Ligands
  • Linear Models
  • Nucleic Acid Hybridization / methods*
  • Oligonucleotides / chemistry
  • Oligonucleotides / genetics
  • Oligonucleotides / metabolism*
  • Oligonucleotides / therapeutic use*
  • Polymerase Chain Reaction
  • Polynucleotide 5'-Hydroxyl-Kinase / metabolism
  • Reproducibility of Results

Substances

  • Ligands
  • Oligonucleotides
  • Polynucleotide 5'-Hydroxyl-Kinase
  • DNA Ligases