Brightness through Local Constraint-LNA-Enhanced FIT Hybridization Probes for In Vivo Ribonucleotide Particle Tracking
Brightness through Local Constraint-LNA-Enhanced FIT Hybridization Probes for In Vivo Ribonucleotide Particle Tracking
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DOI：
10.1002/anie.201406022
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发表时间：
2014-10-13
影响因子：
16.6
通讯作者：
Seitz, Oliver
中科院分区：
文献类型：
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作者：
Hoevelmann, Felix;Gaspar, Imre;Seitz, Oliver
对活细胞中的RNA动态进行成像通常是通过转基因方法来实现的，这种方法需要对RNA靶标和细胞进行修饰。荧光杂交探针也将允许对野生生物进行分析。我们开发了抗核酸酶的DNA强制插层(FIT)探针，它结合了杂交时荧光的高度增强和跟踪单个核糖核苷酸颗粒(RNP)所需的高亮度。在我们的设计中，一个单一的噻唑橙(TO)插入剂染料被连接作为碱基替代品，而相邻的锁定核酸(LNA)单元用于引入局部约束。这关闭了荧光衰减通道，从而增加了探针-目标双链的亮度。只要两个探针就足以在野生黑腹果蝇卵母细胞中追踪Oskar mRNPs。
Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild-type organisms. We developed nuclease-resistant DNA forced intercalation (FIT) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic acid (LNA) unit serves to introduce a local constraint. This closes fluorescence decay channels and thereby increases the brightness of the probe-target duplexes. As few as two probes were sufficient to enable the tracking of oskar mRNPs in wild-type living Drosophila melanogaster oocytes.