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Tritium labeling of full length small interfering RNA’s

Christensen, Jesper and Natt, Francois and Hunziker, Juerg and Krauser, Joel and Andres, Hendrik and Swart, Pieter Jacob (2012) Tritium labeling of full length small interfering RNA’s. Journal of Labelled Compounds and Radiopharmaceuticals, 55 (6). pp. 189-196. ISSN 0362-4803

Abstract

A simple procedure is described for full length single internal [3H]-radiolabeling of oligonucleotides. Previous labeling strategies have been applied to large molecular weight compounds like proteins and oligonucleotides, e.g. iodination, and 111In labeling via covalently bounded chelators. However, a procedure has not yet been reported for single internal radiolabeling of oligonucleotides that preserves the molecular structure (3H replacing a 1H). Following our strategy the radiolabel can be strategically placed within a stable and predetermined internal position of the siRNA. This placement was accomplished by placing a 5-bromouridine or 5-bromo-2´-O-methyluridine phosphoramidite building block into the middle of the antisense strand using standard phosphoramidite chemistry. The de-protected full length antisense strand was tritium labeled by bromine/tritium exchange, catalyzed by palladium on charcoal in the predetermined 5-position of either uridine or 2´-O-methyluridine. Internal placement of the building block within the oligonucleotide sequence and label placement at 5-position decreases the likelihood of the label to be readily cleaved from the oligonucleotide in vivo, and loss of the label by spontaneous tritium-hydrogen exchange. The tritiated single stranded and double stranded RNAs were also shown to both radio and chemically stable for at least 6 months at -80ºC. This allows more than sufficient time to conduct pharmaceutical formulation and pharmacokinetic studies.

Item Type: Article
Additional Information: Work has already been presented as a poster at an international conference (7th Oligonucleotide Therapeutics Society, Copenhagen September 2011). AOK ID 5796 (and 5076). Patentability has previosuly been discussed and declined by Kristin Konzak and Heather Woodcock-Bourne from IP. This work is part of my PhD project.
Keywords: oligonucleotides; siRNA; tritiation; halogen-tritium exchange; final step radiolabeling
Date Deposited: 13 Oct 2015 13:14
Last Modified: 13 Oct 2015 13:14
URI: https://oak.novartis.com/id/eprint/6594

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