Degradation of Nuclease-Stabilized RNA Oligonucleotides in Mycoplasma-Contaminated Cell Culture Media
Degradation of Nuclease-Stabilized RNA Oligonucleotides in Mycoplasma-Contaminated Cell Culture Media
复制标题
DOI：
10.1089/nat.2011.0316
复制
发表时间：
2012-02-01
影响因子：
4
通讯作者：
McNamara, James O., II
中科院分区：
文献类型：
--
作者：
Hernandez, Frank J.;Stockdale, Katie R.;McNamara, James O., II
人工RNA试剂如小干扰RNA（siRNA）和适体通常必须进行化学修饰，以在包括核糖核酸酶的环境中获得最佳效果。支原体是哺乳动物细胞培养物的常见细菌污染物，已知其产生核糖核酸酶。在这里，我们描述了快速降解的核酸酶稳定的RNA寡核苷酸在人胚肾293（HEK）细胞培养物与支原体发酵，一种常见的支原体污染。具有2 '-氟-或2' 0-甲基-修饰的嘧啶的RNA在来自该培养物的条件培养基中容易降解，但在来自未污染的HEK细胞的条件培养基中稳定。用2 '-O-甲基完全修饰的RNA在支原体污染的培养基中不降解。条件培养基和从培养基离心的材料的RNA酶谱分析揭示了几个不同的蛋白质条带（范围从30至68 kDa），其能够用2 '-氟-或2'-O-甲基-修饰的嘧啶降解RNA。最后，在从污染的培养物上清液中离心的材料中检测到支原体相关核酸酶，所述材料用含有RNA寡核苷酸的2 '-O-甲基修饰的嘧啶在短至15分钟内检测到，并且用5'-荧光素酰胺（FAM）和3 '-猝灭剂标记。这些结果表明，支原体污染可能是涉及基于RNA的试剂的细胞培养实验的关键混杂变量，与涉及裸RNA的应用特别相关（例如，适体-siRNA嵌合体）。
Artificial RNA reagents such as small interfering RNAs (siRNAs) and aptamers often must be chemically modified for optimal effectiveness in environments that include ribonucleases. Mycoplasmas are common bacterial contaminants of mammalian cell cultures that are known to produce ribonucleases. Here we describe the rapid degradation of nuclease-stabilized RNA oligonucleotides in a human embryonic kidney 293 (HEK) cell culture contaminated with Mycoplasma fermentans, a common species of mycoplasma. RNA with 2'-fluoro- or 2'O-methyl-modified pyrimidines was readily degraded in conditioned media from this culture, but was stable in conditioned media from uncontaminated HEK cells. RNA completely modified with 2'-O-methyls was not degraded in the mycoplasma-contaminated media. RNA zymogram analysis of conditioned culture media and material centrifuged from the media revealed several distinct protein bands (ranging from 30 to 68 kDa) capable of degrading RNA with 2'-fluoro- or 2'-O-methyl-modified pyrimidines. Finally, the mycoplasma-associated nuclease was detected in material centrifuged from the contaminated culture supernatants in as little as 15 minutes with an RNA oligo-containing 2'-O-methyl-modified pyrimidines and labeled with a 5'-fluorescein amidite (FAM) and 3'-quencher. These results suggest that mycoplasma contamination may be a critical confounding variable for cell culture experiments involving RNA-based reagents, with particular relevance for applications involving naked RNA (e.g., aptamer-siRNA chimeras).