Tritium labeling of full‐length small interfering RNAs

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 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 °C. This allows more than sufficient time to conduct pharmaceutical formulation and pharmacokinetic studies. Copyright © 2012 John Wiley & Sons, Ltd.

Metabolism Studies of Unformulated Internally [3H]-Labeled Short Interfering RNAs in Mice

“…A novel 3 H-radiolabeling method of oligonucleotides (Christensen et al, 2012) was used to perform preclinical ADME studies of unformulated siRNAs. Tritiated oligonucleotides offer several advantages for ADME studies, including ]siRNAs have been proven to be both radio and chemically stable for at least 6 months at 280°C (Christensen et al, 2012), which allows a practical time window to conduct pharmaceutical formulation and pharmacokinetic studies; (3) the 3 H-label is not released by metabolic processes from the guide strand of the siRNA until it has been extensively degraded and is likely then to be RNA interference inactive; (4) simplification of the analysis of metabolites because of the presence of a single radiolabeled site in the oligonucleotide; and (5) the method is solely dependent on the existence of a uridine or a 2´-O-methyluridine residue in the sequence. This method should be applicable to chemically modified and unmodified phosphodiester oligonucleotides.…”

“…For this purpose, the siRNAs were internally labeled with tritium according to procedures previously described (Christensen et al, 2012). The MRP4 (multidrug resistance protein isoform 4) siRNA targets the transporter protein, MRP4, which is expressed in the brush-border membrane of the rat kidney.…”

“…For our work, a method for labeling siRNAs with tritium at a single internal nucleotide position (uridine or 2´-O-methyluridine) was applied (Christensen et al, 2012). The method is thought to eliminate the previously described issues and, therefore, provides a suitable research tool for absorption, distribution, metabolism, and excretion (ADME) studies of siRNAs.…”

Biodistribution and Metabolism Studies of Lipid Nanoparticle–Formulated Internally [3H]-Labeled siRNA in Mice

“…The siRNA was internally labeled with tritium following procedures previously described (Christensen et al, 2012). The 21mer double-stranded sequences were as follows: 59-UuAcAUu 7 AAAGUCUGUUGUuu-39 and 59-AcAAcAGAcuuuAAuGuAAuu-39 for the guide and passenger strands, respectively.…”

“…For our work, we chose the SSB siRNA which targets the ubiquitous gene Sjögren syndrome antigen B (SSB). Therefore, we applied a single internal [ 3 H]-radiolabeling procedure, in which the full-length oligonucleotides were radiolabeled by Br/ 3 H exchange (Christensen et al, 2012). This labeling method avoids chemical modification of the siRNA by placing the tritium atom in a chemically stable and predetermined internal position.…”