Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Cell Biology and Physiology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
PLoS One. 2014 Jan 15;9(1):e85813. doi: 10.1371/journal.pone.0085813. eCollection 2014.
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.
由于 RNA 的时空和亚细胞加工，RNA 表达与细胞功能之间的关系通常难以破译。当仅获取 RNA 表达的全局测量值时，这些 RNA 调控的复杂性常常被忽视。这导致了几种工具的发展，这些工具允许在活细胞中实时成像单个工程化的 RNA 转录本。在这里，我们描述了一种新的技术，该技术利用基于寡核苷酸的探针，即比率双分子信标（RBMB），来成像工程化的 RNA 转录本，这些转录本在 3'-非翻译区中包含 96 个串联重复的 RBMB 靶序列。RBMB 与靶 RNA 的结合导致离散的亮荧光点，代表单个转录本，可以实时成像。由于 RBMB 是一种合成探针，因此使用光稳定、明亮且红移的荧光团导致高信号背景比。通过均方位移和矩标度谱分析很容易对 RNA 运动进行特征化。这些分析揭示了定向、布朗和亚扩散运动的清晰示例。
