Tritium labeling of antisense oligonucleotides by exchange with tritiated water

• Published in: Nucleic acids research 1993-08, Vol.21 (16), p.3737-3743
• Main Authors: Graham, M.J., Freier, S.M., Crooke, R.M., Ecker, D.J., maslova, R.N., Lesnik, E.a.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological and medical sciences; General pharmacology; HeLa Cells; Humans; Kinetics; Medical sciences; Mercaptoethanol; Methods; Mice; Molecular Sequence Data; Oligonucleotides, Antisense - chemistry; Oligonucleotides, Antisense - metabolism; Pharmacokinetics. Pharmacogenetics. Drug-receptor interactions; Pharmacology. Drug treatments; Thermodynamics; Tritium; Tumor Cells, Cultured; Water - chemistry
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/21.16.3737

We describe a simple, efficient, procedure for labeling oligonucleotides to high specific activity (> 1×108 cpm/μmol) by hydrogen exchange with tritlated water at the C8 positions of purines in the presence of β-mercaptoethanol, an effective radical scavenger. Approximately 90% of the starting material is recovered as intact, labeled oligonucleotide. The radiolabeled compounds are stable in biological systems; greater than 90% of the specific activity is retained after 72 hr incubation at 37°C in serum-containing media. Data obtained from in vitro cellular uptake experiments using oligonucleotides labeled by this method are similar to those obtained using 35S or 14C-labeled compounds. Because this protocol is solely dependent upon the existence of purine residues, it should be useful for radiolabeling modified as well as unmodified phosphodlester oligonucleotides.