Imaging the directed transport of single engineered RNA transcripts in real-time using ratiometric bimolecular beacons.
Imaging the directed transport of single engineered RNA transcripts in real-time using ratiometric bimolecular beacons.
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DOI：
10.1371/journal.pone.0085813
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Tsourkas A
中科院分区：
文献类型：
--
作者：
Zhang X;Zajac AL;Huang L;Behlke MA;Tsourkas A
由于RNA的时间加工和亚细胞加工的存在，RNA表达和细胞功能之间的关系往往难以破译。当仅获得RNA表达的全局测量时，这些RNA调控的复杂性往往被忽视。这导致了几种工具的发展，这些工具可以实时成像活细胞中单个工程RNA转录物。在这里，我们描述了一种新技术，利用基于寡核苷酸的探针，比率双分子信标（RBMB），来成像RNA转录本，这些转录本被设计成在3 ' -非翻译区包含96个RBMB目标序列的串联重复序列。RBMBs与靶RNA结合产生离散的明亮荧光点，代表单个转录本，可以实时成像。由于RBMBs是一种合成探针，使用光稳定，明亮和红移的荧光团导致高信号对背景。RNA的运动很容易被均方位移和矩标度谱分析表征。这些分析揭示了定向运动、布朗运动和亚扩散运动的清晰例子。
The relationship between RNA expression and cell function can often be difficult to decipher due to the presence of both temporal and sub-cellular processing of RNA. These intricacies of RNA regulation can often be overlooked when only acquiring global measurements of RNA expression. This has led to development of several tools that allow for the real-time imaging of individual engineered RNA transcripts in living cells. Here, we describe a new technique that utilizes an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), to image RNA transcripts that were engineered to contain 96-tandem repeats of the RBMB target sequence in the 3′-untranslated region. Binding of RBMBs to the target RNA resulted in discrete bright fluorescent spots, representing individual transcripts, that could be imaged in real-time. Since RBMBs are a synthetic probe, the use of photostable, bright, and red-shifted fluorophores led to a high signal-to-background. RNA motion was readily characterized by both mean squared displacement and moment scaling spectrum analyses. These analyses revealed clear examples of directed, Brownian, and subdiffusive movements.
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影响因子：
14.9
作者：
Chen AK;Behlke MA;Tsourkas A
通讯作者：
Tsourkas A
影响因子：
10.8
作者：
Kang W;Yavari F;Minary-Jolandan M;Giraldo-Vela JP;Safi A;McNaughton RL;Parpoil V;Espinosa HD
通讯作者：
Espinosa HD
影响因子：
64.5
作者：
Cha, BJ;Koppetsch, BS;Theurkauf, WE
通讯作者：
Theurkauf, WE
影响因子：
9.2
作者：
Forrest, KM;Gavis, ER
通讯作者：
Gavis, ER
DOI：
10.1073/pnas.2233244100
发表时间：
2003-11-11
影响因子：
11.1
作者：
Bratu, DP;Cha, BJ;Tyagi, S
通讯作者：
Tyagi, S