Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls

Abstract

Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, 'experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls.

From epigenetics to anti-doping application: a new tool of detection

“…Since the real problem is to discriminate between endogenous and exogenous GH, it is used to detect the abuse by an indirect method based on the evaluation of IGF-1 level [67]. All the strategies applied to characterize siRNA in biological fluids are comparable to an experimental 'RNomics' approach, which may help as a potential link between genome and proteome in the next future [68]. Also called antagomirs, they represent a new strategy that could play an important role in doping control.…”

Human sports drug testing by mass spectrometry

“…In order to study the fitness‐for‐purpose of doping control analytical methods for targeting siRNA and potential metabolites after i.v . administration, animal elimination studies were conducted with model compounds (Thomas et al, ). Up to 36 hr post‐application of the drug, plasma and urine was collected from laboratory rodents and specimens were subjected to RNA‐specific purification strategies prior to gel electrophoretic and LC‐MS/MS analysis.…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…Due to the fact that siRNA‐based drugs predominantly require stabilizing modifications, for example 2'‐O‐methylation or the use of locked nucleic acids, non‐natural targets are provided for sports drug testing purposes. To probe for the metabolic fate of intravenously administered siRNA and the possibility to detect the intact substance as well as metabolites in urine, animal studies were conducted with model siRNA designed to interfere with myostatin . Using conventional RNA extraction kits, LC‐HRMS top‐down as well as bottom‐up, and alternatively SDS‐PAGE analyses, the model substances and metabolites were detected in rat urine for up to 24 h following a single dose of siRNA administered intravenously at 1 mg/kg.…”