Christensen, Jesper, Natt, Francois, Hunziker, Juerg, Krauser, Joel, 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