Tritium labeling of full‐length small interfering RNAs

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 such as proteins and oligonucleotides, for example, 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). 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?degrees C. This allows more than sufficient time to conduct pharmaceutical formulation and pharmacokinetic studies. Copyright (c) 2012 John Wiley & Sons, Ltd.