生命的化学2023,Vol.43Issue(12) :1963-1971.DOI:10.13488/j.smhx.20230618

siRNA储存有效期的多维度分析

Multidimensional analysis of the shelf life of siRNA storage

李萍 蒙羽涵 徐维勇 黄永奎 蒋素梅 黄艳娜 唐雪明
生命的化学2023,Vol.43Issue(12) :1963-1971.DOI:10.13488/j.smhx.20230618

siRNA储存有效期的多维度分析

Multidimensional analysis of the shelf life of siRNA storage

李萍 1蒙羽涵 1徐维勇 1黄永奎 1蒋素梅 1黄艳娜 1唐雪明1
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作者信息

  • 1. 硅羿科技(上海)有限公司,上海 201300
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摘要

小干扰RNA(small interfering RNA,siRNA)药物研究方兴未艾.本研究为探讨不同的化学修饰和存储方式对siRNA储存有效期的影响,裸序列(siRNA-G)、硫代修饰(siRNA-T1)和组合修饰(硫代+锁核苷酸+甲氧基:siRNA-M8)的siRNA,被分别应用3种方式储存,即-20℃原液、-20℃冻干粉及室温冻干粉.不同的检测结果表明,-20℃原液最佳有效期是60 d;-20℃冻干粉和室温冻干粉最佳有效期分别是120 d和60 d;修饰方式对储存有效期影响小.采用高效液相色谱法(high performance liquid chromatography,HPLC)和气相色谱法(gas chromatography,GC)测得siRNA-G、siRNA-M8及siRNA-T1的纯度分别为98.51%、98.90%和97.20%;在最佳有效期内,马铃薯葡萄糖琼脂培养基(potato dextrose agar,PDA)检测所有形式的siRNA无菌性合格(未长菌);细胞内靶基因敲降水平在有效期内大于35%;熔解温度(melting temperature,Tm)在有效期内稳定(Tm值波动<6%);序列表征基本合格.本研究数据旨在为合理有效使用siRNA提供了重要参考.

Abstract

The research of siRNA drugs is in the ascendant.To investigate the effects of different chemical modifications and storage methods on the validity period of siRNA,unmodified(siRNA-G),thio-modified(siRNA-T1)and combinatorially modified(thio+locked nucleotide+methoxy group:siRNA-M8)siRNAs were stored under three different conditions,namely raw siRNA solution at-20℃,freeze-dried siRNA powder at-20℃or room temperature.The different testing results showed that validity period was 60 days for raw siRNA solution at-20℃,120 and 60 days for freeze-dried siRNA powder at-20℃and room temperature,respectively,while the ways of chemical modifications had neglectful influence on the validity period.The initial purity of siRNA-G,siRNA-M8 and siRNA-T1 are 98.51%,98.90%and 97.20%by HPLC and GC,respectively.During the best valid life,all kinds of siRNA were detected as sterile by potato dextrose agar(PDA)method.The knock down level of intracellular target gene was greater than 35%within valid period.The Tm value is stable during the validity period.The sequence characterization is qualified.The current data provided an important reference for the rational and effective use of siRNA.

关键词

siRNA合成/冻干/储存有效期

Key words

siRNA synthesis/freeze drying/storage validity perio

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出版年

2023
生命的化学
中国生物化学与分子生物学会

生命的化学

CSTPCD
影响因子:0.404
ISSN:1000-1336
参考文献量3
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