A conformation-induced fluorescence method for microRNA detection.
A conformation-induced fluorescence method for microRNA detection.
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DOI：
10.1093/nar/gkw108
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发表时间：
2016-06-02
影响因子：
14.9
通讯作者：
Cohen SM
中科院分区：
文献类型：
--
作者：
Aw SS;Tang MX;Teo YN;Cohen SM
MicroRNA 通过调节靶 mRNA 表达在多种生物系统和过程中发挥重要作用，并显示出作为临床生物标志物的前景。然而，它们的小尺寸给标记或直接检测带来了挑战。传感和量化 microRNA 的技术创新可能有助于研究 microRNA 生物学的新方面，并有助于诊断学的发展。通过在荧光 RNA Spinach 中引入额外的茎环并改变其 3' 和 5' 末端，我们生成了一种新的 RNA，Pandan，它作为 microRNA 传感器的基础。 Pandan 含有两个序列可变的茎环，编码目标 microRNA 的互补序列。在其传感器形式中，它需要结合目标 microRNA，以便重建用于荧光团结合和荧光的 RNA 支架。目标 microRNA 的结合导致荧光强度发生较大变化。所测试传感器观察到的荧光中值倍数变化约为 50 倍。 Pandan RNA 传感器表现出良好的信噪比，并且可以检测复杂 RNA 混合物中的目标 microRNA。
MicroRNAs play important roles in a large variety of biological systems and processes through their regulation of target mRNA expression, and show promise as clinical biomarkers. However, their small size presents challenges for tagging or direct detection. Innovation in techniques to sense and quantify microRNAs may aid research into novel aspects of microRNA biology and contribute to the development of diagnostics. By introducing an additional stem loop into the fluorescent RNA Spinach and altering its 3′ and 5′ ends, we have generated a new RNA, Pandan, that functions as the basis for a microRNA sensor. Pandan contains two sequence-variable stem loops that encode complementary sequence for a target microRNA of interest. In its sensor form, it requires the binding of a target microRNA in order to reconstitute the RNA scaffold for fluorophore binding and fluorescence. Binding of the target microRNA resulted in large changes in fluorescence intensity. The median fold change in fluorescence observed for the sensors tested was ∼50-fold. Pandan RNA sensors exhibit good signal-to-noise ratios, and can detect their target microRNAs within complex RNA mixtures.