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 × 10⁴ (standard deviation: 4.34 × 10³) 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.

中文翻译：

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使用 MALDI-TOF 质谱法测定硫代磷酸化寡核苷酸的序列以控制马术运动中的基因兴奋剂

在人类和马术运动项目中，一种基因掺杂方法是非法使用治疗性寡核苷酸来改变基因表达。在这项研究中，我们旨在通过使用基质辅助激光解吸/电离飞行时间质谱 (MALDI-TOF MS) 进行测序来鉴定治疗性寡核苷酸。作为治疗性寡核苷酸的模型，使用了 22 bp 长的硫代磷酸化寡核苷酸 (PSO)。^{通过使用 Clarity OTX 试剂盒提取短长度寡核苷酸，得到平均强度为 6.08 × 10 4}的单电荷 PSO 光谱（标准偏差：4.34 × 10 ³) 使用 MALDI-TOF MS 的线性负模式从 1 ml 马血浆中的 500 pmol PSO 中检测到。此外，使用源内衰减 (ISD) 模式确定 17 bp 序列，表明 1 ml 血浆中 500 pmol PSO 是测序的检测限。使用确定的序列 (17 bp)，通过 GGGenome 搜索在马参考基因组 EquCab2.0 上单独鉴定了 PSO 的靶向基因。这些程序可潜在地用于识别核苷酸未知的治疗性寡核苷酸，用于基因兴奋剂控制。