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[目的]以microRNA-21(miR-21)为靶点,探索制备放射性核素锝[99mTc]标记的反义寡核苷酸探针的方法与实验条件。[方法]设计化学结构修饰的反义寡核苷酸序列并进行合成,利用双功能螯合剂NHS-MAG3偶联待标记探针和99mTc,优化不同实验条件下的标记率及放化纯,并对标记产物进行凝胶电泳鉴定及稳定性评价。[结果]降低氯化亚锡的用量、减少99mTc的反应体积、适度延长标记反应时间均可显著提高标记率,最高可达97%。标记探针在血清37℃条件中孵育6h其放化纯仍可维持在96%以上。凝胶电泳结果证实核酸条带与放射性分布一致。[结论]通过优化探针设计和标记反应条件能够大大提高标记率和放化纯度,无需纯化即可进一步应用,达到简化制备程序的目的。
[Objective] To explore the method and experimental conditions for preparing antisense oligonucleotide probes labeled with radionuclide technetium [99mTc] using microRNA-21 (miR-21) as a target. [Method] The antisense oligonucleotide sequence modified by chemical structure was designed and synthesized. The dual-functional chelator NHS-MAG3 was used to couple the labeled probe and 99mTc. The labeling rate and radiochemical purity under different experimental conditions were optimized. The labeled product was identified by gel electrophoresis and its stability was evaluated. [Result] Reducing the amount of stannous chloride, reducing the reaction volume of 99mTc, and prolonging the labeling reaction time could significantly increase the labeling rate up to 97%. The labeled probe could still maintain more than 96% of radiochemical purity when incubated in serum at 37 ℃ for 6h. Gel electrophoresis results confirmed that the nucleic acid bands and radioactive distribution. [Conclusion] The labeling rate and radiochemical purity can be greatly improved by optimizing the probe design and labeling reaction conditions, and can be further applied without purification to achieve the purpose of simplifying the preparation procedure.