Fluorogenic RNA nanoparticles for monitoring RNA folding and degradation in real time in living cells.
Fluorogenic RNA nanoparticles for monitoring RNA folding and degradation in real time in living cells.
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DOI：
10.1089/nat.2012.0380
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发表时间：
2012-11
影响因子：
4
通讯作者：
Randall D. Reif;Farzin Haque;Peixuan Guo
中科院分区：
文献类型：
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作者：
Randall D. Reif;Farzin Haque;Peixuan Guo
随着细胞非编码RNA功能的发现，将小干扰RNA（siRNA）、微小RNA（miRNA）、核酶、核糖开关等RNA导入细胞调节细胞生命周期和治疗疾病的方法已成为常规。了解RNA进入细胞后的折叠、降解和细胞内半衰期是生物学和药理学中一个有趣的问题。目前，在细胞内真实的时间内检测RNA折叠、降解和半衰期的方法极具挑战性。测量体内RNA半衰期和降解的常用测定方法是使用放射性标记物或荧光RNA标记。挑战在于，在RNA被降解或错误折叠后，同位素或荧光仍然存在于细胞中，因此信号不是细胞中RNA存在的真实指示。通常用于测量RNA寿命的替代方法是从细胞中分离RNA，并通过凝胶，色谱或毛细管电泳区分完整和降解的RNA。然而，当细胞分解时，核糖核酸酶（RNase）将从细胞区室释放，并且细胞裂解物中的小RNA的降解在细胞裂解后立即发生。在这里，我们报道了一种使用荧光RNA与RNA纳米技术组合来监测活细胞中真实的RNA降解的方法（Guo，2010; Guo等人，2012年）。将结合孔雀石绿色（MG）的RNA适体、切割肝炎病毒基因组的核酶和萤火虫荧光素酶的siRNA都融合到噬菌体phi 29包装RNA（pRNA）3-way junction（3 WJ）基序以产生RNA纳米颗粒。MG适体、B型肝炎病毒核酶和荧光素酶siRNA在融合到纳米颗粒中后都独立地保留了它们的功能。当RNA纳米颗粒降解、变性或错误折叠时，荧光消失。MG本身不发荧光，只有当RNA保持正确构象折叠时，它才能与其适体结合并发射荧光。因此，MG适体荧光（在MG染料存在下）可用作使用落射荧光显微镜和荧光光谱法在不裂解细胞的情况下真实的时间内对细胞中RNA纳米颗粒、siRNA、适体和核酶的降解和折叠的量度。我们表明，电穿孔的含有MG适体的RNA纳米颗粒的半衰期（t1/2）在电穿孔到细胞中后为4.3小时。
Due to the discovery of more and more roles of cellular noncoding RNAs, the approaches for introducing RNAs including small interfering RNA (siRNA), micro RNA (miRNA), ribozyme, and riboswitch into cells for regulating cell life cycle and for the treatment of diseases have become routine practice. The understanding of RNA folding, degradation, and intracellular half-life after entering the cell is an intriguing question in biology and pharmacology. Currently, methods to detect RNA folding, degradation, and half-life in real time within the cell is extremely challenging. The common assay method to measure RNA half-life and degradation in vivo is the use of radioactive markers or fluorescence RNA labeling. The challenge is, after RNA becomes degraded or misfolded, the isotope or the fluorescence is still present in the cell, thus the signals are not a true indication of the presence of the RNA in the cell. The alternate method commonly used to measure RNA life is to isolate RNA from cells and distinguish between intact and degraded RNA by gel, chromatography, or capillary electrophoresis. However, when a cell is breaking down, ribonucleases (RNases) will be released from cell compartments, and degradation of small RNA in cell lysates occurs immediately after cell lysis. Here we report a method to monitor RNA degradation in real time in living cells using fluorogenic RNA in combination with RNA nanotechnology (Guo, 2010; Guo et al., 2012). The RNA aptamer that binds malachite green (MG), the ribozyme that cleaves the hepatitis virus genome, and a siRNA for firefly luciferase were all fused to the bacteriophage phi29 packaging RNA (pRNA) 3-way junction (3WJ) motif to generate RNA nanoparticles. The MG aptamer, the hepatitis B virus ribozyme, and the luciferase siRNA all retained their function independently after fusion into the nanoparticles. When the RNA nanoparticle is degraded, denatured, or misfolded, the fluorescence disappears. MG, which is not fluorescent by itself, is capable of binding to its aptamer and emitting fluorescent light only if the RNA remains folded in the correct conformation. Therefore, the MG aptamer fluorescence (in the presence of MG dye) can be used as a measure of the degradation and folding of RNA nanoparticles, the siRNA, the aptamer, and the ribozyme in the cell in real time using epifluorescence microscopy and fluorescence spectroscopy without lysing the cells. We show that the half-life (t½) of the electroporated MG aptamer containing RNA nanoparticle was 4.3 hours after electroporation into cells.