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Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports.

Authors :
Tozaki T
Kwak HG
Nakamura K
Takasu M
Ishii H
Ohnuma A
Kikuchi M
Ishige T
Kakoi H
Hirota KI
Kusano K
Hirata M
Nirasawa T
Nagata SI
Source :
Drug testing and analysis [Drug Test Anal] 2022 Jan; Vol. 14 (1), pp. 175-180. Date of Electronic Publication: 2021 Sep 08.
Publication Year :
2022

Abstract

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 10 <superscript>4</superscript> (standard deviation: 4.34 × 10 <superscript>3</superscript> ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control.<br /> (© 2021 John Wiley & Sons, Ltd.)

Details

Language :
English
ISSN :
1942-7611
Volume :
14
Issue :
1
Database :
MEDLINE
Journal :
Drug testing and analysis
Publication Type :
Academic Journal
Accession number :
34418319
Full Text :
https://doi.org/10.1002/dta.3154