RNA interference for performance enhancement and detection in doping control

Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

doi:10.1002/dta.330

Cited by 11 publications

(12 citation statements)

References 43 publications

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“…[27,28] This strategy is comparable to an 'experimental RNomics' approach which may bear the potential to experimentally link the genome to the proteome in the near future. Besides the identification of modified nucleotides by LC-HRMS after alkaline hydrolysis, also the intact RNA respectively their truncated metabolites were determined under consideration of the accurate masses of the multiply negatively charged sense and antisense strand ions in the mass spectrum.…”

Section: Resultsmentioning

confidence: 99%

“…The subsequent identification of the characteristic RNA fragments by LC-HRMS provides a sophisticated tool to identify also unknown siRNAs from biological fluids, which are potentially used as gene doping agents. [27,28] This strategy is comparable to an 'experimental RNomics' approach which may bear the potential to experimentally link the genome to the proteome in the near future. The complete workflow developed within this project is summarized in Figure 6.…”

Section: Resultsmentioning

confidence: 99%

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

Thomas

Walpurgis

Delahaut

et al. 2013

Drug Testing and Analysis

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Thomas

Walpurgis

Delahaut

et al. 2013

Drug Testing and Analysis

Self Cite

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“…One of the most prominent examples is represented by the myostatin gene, which currently has the highest potential for misuse in sport, given the numerous investigations in drug research. The downregulation of this gene has been shown to induce an improvement in performance and an increase in muscle mass [69].…”

Section: Biomarkers For Detection Of Doping In Sportmentioning

confidence: 99%

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

Schiera¹,

Contrò²,

Sacco³

et al. 2018

Hum Mov.

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Eukaryotic genomes transcribe up to 90% of the genomic DNA but only 1-2% of these transcripts encode for proteins, whereas the vast majority are transcribed as non-coding RNAs (ncRNAs). They are divided into short ncRNA, particularly micro-RNA (miRNA) and small interference RNA (siRNA), and long ncRNAs. Noteworthy, they are unexpectedly stable since they are protected from degradation through different mechanisms: package in exosomes/microvesicles structures, in apoptotic bodies, in HDL lipoprotein, or by RNA binding proteins. For several years already, biomarkers have been used to detect biological disease; in the last years, a requirement appeared to find some of them to unearth the signs of doping. The potential of ncRNAs as a biological candidate is strongly debated and it seems to have become the right tool in the anti-doping hands. In the recent years, the next-generation sequencing (NGS) technology was used by the World Anti-Doping Agency to draft the athlete biological passport (ABP), measuring the circulating miRNAs and applying these new biomarkers in anti-doping. NGS technology does not require any prior knowledge of ncRNAs, but the limit to employ this biomarker to detect performanceenhancing drug use must consider the intrinsic and extrinsic factors that might affect measurements.

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“…Two major scenarios of gene doping are described with one being the abuse of ‘classical’ gene therapy, i.e. introduction of synthetic DNA sequences via viral vehicles into the organism, and the other being based on RNA interference strategies . The latter has recently been considered the more promising approach in therapeutic settings, which however also implies that there is a higher risk of its abuse in sports.…”

Section: Gene Dopingmentioning

confidence: 99%

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

Thevis¹,

Kuuranne

Geyer

et al. 2012

Drug Testing and Analysis

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Frequently updated anti-doping regulations and detection strategies for banned substances and methods of doping are vital for efficient and scientifically substantiated abatement of drug abuse, manipulation, and illicit performance enhancement in sport. In 2012, the ninth version of the Prohibited List as annually issued by the World Anti-Doping Agency (WADA) reports on modest but relevant modifications from its 2011 predecessor. Awareness of old and new analytical challenges in sports drug testing, together with issues resulting from altered rules and regulations, have given rise to numerous research projects aiming at improved detection strategies to strengthen international anti-doping efforts. In this annual banned substance review, emerging and advancing methods in the detection of known and recently outlawed substances are reported. New and/or enhanced procedures and techniques of doping analysis are reviewed together with information relevant to doping controls. The review surveys and critiques literature published between October 2011 and September 2012.

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RNA interference for performance enhancement and detection in doping control

Cited by 11 publications

References 43 publications

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

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

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

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

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