A convenient thiazole orange fluorescence assay for the evaluation of DNA duplex hybridization stability.
A convenient thiazole orange fluorescence assay for the evaluation of DNA duplex hybridization stability.
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一种方便的噻唑橙荧光测定法，用于评估 DNA 双链体杂交稳定性。
DOI：
10.1007/s11307-009-0221-4
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发表时间：
2009
影响因子：
3.1
通讯作者：
Hnatowich,DonaldJ
中科院分区：
文献类型：
--
作者：
Liang,Minmin;Liu,Xinrong;Nakamura,Kayoko;Chen,Xiangji;Cheng,Dengfeng;Liu,Guozheng;Dou,Shuping;Wang,Yi;Rusckowski,Mary;Hnatowich,DonaldJ
本文介绍了一种简便、快速测定DNA双链体和其它寡聚体在不同环境中杂交稳定性的方法。当加入到低聚物双链体中时，噻唑橙子（TO）染料嵌入，并且在这种状态下是荧光的。因此，当双链体解离发生时，TO的释放导致荧光强度的可检测变化。方法本研究的两个反义寡聚体均为25聚体的硫代磷酸（PS）DNA，一个针对RIα mRNA，另一个针对mdr 1 mRNA。前一种双链体首先用于溶液研究，在大多数情况下与16聚体磷酸二酯（PO）互补DNA（即，PS-DNA25/PO-cDNA16）。两个双链体然后在一系列细胞研究中使用SK BR 3（RIα+）、KB G2（mdr 1 ++）和KB 31（mdr 1 +/-）cells.ResultsPreliminary measurements in solution显示当超过10个TO分子结合到每个双链体时达到最大荧光。当加入具有RIα mRNA碱基序列的25 mer PO-DNA或PO-RNA时，随后荧光强度的显著变化表明反义DNA从研究双链体上解离并与靶DNA重新结合。动力学测试表明，该过程在约3分钟内完成。在37°C下与抗RI α研究双链体孵育的SK-BR-3（RIα+）细胞随时间推移的荧光测量显示，在研究双链体解离（可能通过反义机制）导致的荧光损失开始超过持续细胞蓄积导致的荧光增加时达到最大值。在KB-G2（多药耐药1+）细胞孵育的抗-mdr 1 study duplex.ConclusionsWhen研究双链体显示是稳定的，在血清中孵育与他们的靶细胞，该试验成功地检测到解离的证据，最有可能的反义机制。因此，已经开发了能够检测DNA双链体解离的TO荧光测定。
ObjectiveA simple and rapid method for measuring the hybridization stability of duplexes of DNAs and other oligomers in different environments is described. When added to an oligomer duplex, the thiazole orange (TO) dye intercalates and in this state is fluorescent. Therefore, when duplex dissociation occurs, the release of TO results in a detectable change in fluorescence intensity. This assay was developed primarily to screen antisense oligomer duplexes that are stable in serum and in the cytoplasm but unstable in the presence of their target messenger RNA (mRNA).MethodsThe two antisense oligomers of this investigation were both 25 mer phosphorothioate (PS) DNAs, one directed against the RIα mRNA and the other directed against the mdr1 mRNA. The former duplex was first used in the solution studies, in most cases duplexed with a 16 mer phosphodiester (PO) complementary DNA (i.e., PS-DNA25/PO-cDNA16). Both duplexes were then tested in a series of cell studies using SK-BR-3 (RIα+), KB-G2 (mdr1++), and KB-31 (mdr1+/–) cells.ResultsPreliminary measurements in solution showed that maximum fluorescence was achieved when more than ten TO molecules were bound to each duplex. When a 25 mer PO-DNA or PO-RNA with the base sequence of the RIα mRNA was added, the dramatic change in fluorescence intensity that followed signified dissociation of the antisense DNA from the study duplex and reassociation with the target DNA. Kinetic measurements showed that this process was completed in about 3 min. Fluorescent measurements of SK-BR-3 (RIα+) cells incubated at 37°C with the anti-RIα study duplex over time showed a maximum at the point where the loss of fluorescence due to dissociation of the study duplex, probably by an antisense mechanism, began to dominate over the increasing fluorescence due to continuing cellular accumulation. A similar result was observed in the KB-G2 (mdr1+) cells incubated with the anti-mdr1 study duplex.ConclusionsWhen study duplexes shown to be stable in serum were incubated with their target cells, the assay successfully detected evidence of dissociation, most likely by an antisense mechanism. Thus, a TO fluorescence assay has been developed that is capable of detecting the dissociation of DNA duplexes.