Tiny molecular beacons: LNA/2'-O-methyl RNA chimeric probes for imaging dynamic mRNA processes in living cells.
Tiny molecular beacons: LNA/2'-O-methyl RNA chimeric probes for imaging dynamic mRNA processes in living cells.
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DOI：
10.1021/cb300178a
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发表时间：
2012-09-21
影响因子：
4
通讯作者：
Bratu, Diana P.
中科院分区：
文献类型：
--
作者：
Catrina, Irina E.;Marras, Salvatore A. E.;Bratu, Diana P.
用于成像涉及活细胞中RNA的动态过程的新方法正在不断开发和优化。使用从2 '-O-甲基核糖核苷酸（其对细胞核酸酶具有抗性）合成的分子信标是用于真实的时间可视化天然mRNA的既定方法。为了在空间和时间上解析涉及细胞中RNA的动态步骤，分子信标需要有效地与其RNA靶杂交。为了扩大分子信标可访问的靶位点的库，我们减少了它们的探针序列的长度，并通过包含LNA（锁核酸）核苷酸来改变它们的骨架。我们将这些新的LNA/2 '-O-甲基RNA嵌合寡核苷酸命名为“微小分子信标”。我们分析了这些微小的分子信标，发现只需掺入几个LNA核苷酸，这些较短的探针就能稳定地与RNA的结构化区域退火，这比传统分子信标的效果要好。微小分子信标的合成的容易性以及将它们与大量荧光团和猝灭剂偶联的灵活性使得它们对于检测不太丰富和/或高度结构化的RNA是最佳的。为了确定它们在活体标本中检测内源性mRNA的效率，我们设计了对oskar mRNA特异性的微小分子信标，并将其显微注射到活体果蝇卵母细胞中。然后，我们通过旋转圆盘共聚焦显微镜成像活卵母细胞。结果表明，微小的分子信标以比经典设计的分子信标更快的速度与靶mRNA杂交，并且能够进入以前无法进入的靶区域。
New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2'-O-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time. In order to spatially and temporally resolve dynamic steps involving RNA in cells, molecular beacons need to efficiently hybridize to their RNA targets. To expand the repertoire of target sites accessible to molecular beacons, we decreased the length of their probe sequences and altered their backbone by the inclusion of LNA (locked nucleic acid) nucleotides. We named these new LNA/2'-O-methyl RNA chimera oligonucleotides, “tiny molecular beacons”. We analyzed these tiny molecular beacons and found that the incorporation of just a few LNA nucleotides enables these shorter probes to stably anneal to more structured regions of the RNA than is possible with conventional molecular beacons. The ease of synthesis of tiny molecular beacons, and the flexibility to couple them to a large variety of fluorophores and quenchers, renders them optimal for the detection of less abundant and/or highly structured RNAs. To determine their efficiency to detect endogenous mRNAs in live specimens, we designed tiny molecular beacons that were specific for oskar mRNA and microinjected them into living Drosophila melanogaster oocytes. We then imaged the live oocytes via spinning disc confocal microscopy. The results demonstrate that tiny molecular beacons hybridize to target mRNA at faster rates than classically designed molecular beacons, and are able to access previously inaccessible target regions.
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影响因子：
64.5
作者：
Cha, BJ;Koppetsch, BS;Theurkauf, WE
通讯作者：
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Glotzer, JB;Saffrich, R;Ephrussi, A
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作者：
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