Tritium labeling of full‐length small interfering RNAs

A simple procedure is described for full‐length single internal [ 3 H]‐radiolabeling of oligonucleotides. Previous labeling strategies have been applied to large molecular weight compounds such as proteins and oligonucleotides, for example, iodination and 111 In labeling via covalently bounded chelators. However, a procedure has not yet been reported for single internal radiolabeling of oligonucleotides that preserves the molecular structure ( 3 H replacing a 1 H). In 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 deprotected 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 be radio and chemically stable for at least 6 months at −80 °C. This allows more than sufficient time to conduct pharmaceutical formulation and pharmacokinetic studies. Copyright © 2012 John Wiley & Sons, Ltd.