PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells
PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells
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DOI：
10.1021/bc300249f
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发表时间：
2012-10-01
影响因子：
4.7
通讯作者：
Seitz, Oliver
中科院分区：
文献类型：
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作者：
Kummer, Susann;Knoll, Andrea;Seitz, Oliver
允许RNA成像的荧光杂交探针提供了关于如何在活细胞中合成和运输特定RNA分子的信息。在这项研究中，我们探索了基于肽核酸（PNA）的fitt探针在H1N1甲型流感病毒复制周期中表达的两种不同病毒mRNA分子的同时成像。PNA fit探针是非核苷酸的，非结构化探针，含有单一的不对称菁染料，作为荧光碱基替代物。荧光染料作为一种局部插入探针，通过增强荧光来报道目标DNA/RNA的杂交。虽然多路杂交探针有望促进RNA表达的分析，但目前还没有关于两种不同病毒mRNA靶点的双色成像的报道。在这项工作中，我们开发了一套两种不同颜色的PNA fit探针，允许对编码神经氨酸酶（NA）和基质蛋白1 （M1）的mRNA进行光谱分辨率成像；在甲型流感病毒复制过程中执行不同功能的蛋白质。该探针的特点是具有广泛的适用杂交温度范围。相同的探针序列使活细胞RNA成像（37℃）以及实时PCR测量（60℃退火温度）成为可能。这有助于通过定量（qPCR）和定性（成像）手段对RNA表达进行全面分析。共聚焦激光扫描显微镜显示，病毒rna特异性PNA fit探针既不能染色未感染的细胞，也不能染色被对照病毒感染的细胞。在这项可行性研究中，联合使用不同颜色的PNA fit探针揭示了编码NA或M1的H1N1流感mrna的表达模式存在显著差异。这些实验为PNA fit探针在研究两种mRNA物种的活细胞中mRNA合成的时空进展提供了有用的证据。
Fluorogenic hybridization probes that allow RNA imaging provide information as to how the synthesis and transport of particular RNA molecules is orchestrated in living cells. In this study, we explored the peptide nucleic acid (PNA)-based FIT-probes in the simultaneous imaging of two different viral mRNA molecules expressed during the replication cycle of the H1N1 influenza A virus. PNA FIT-probes are non-nucleotidic, nonstructured probes and contain a single asymmetric cyanine dye which serves as a fluorescent base surrogate. The fluorochrome acts as a local intercalator probe and reports hybridization of target DNA/RNA by enhancement of fluorescence. Though multiplexed hybridization probes are expected to facilitate the analysis of RNA expression, there are no previous reports on the dual color imaging of two different viral mRNA targets. In this work, we developed a set of two differently colored PNA FIT-probes that allow the spectrally resolved imaging of mRNA coding for neuraminidase (NA) and matrix protein 1 (M1); proteins which execute distinct functions during the replication of the influenza A virus. The probes are characterized by a wide range of applicable hybridization temperatures. The same probe sequence enabled live-cell RNA imaging (at 37 degrees C) as well as real-time PCR measurements (at 60 degrees C annealing temperature). This facilitated a comprehensive analysis of RNA expression by quantitative (qPCR) and qualitative (imaging) means. Confocal laser scanning microscopy showed that the viral-RNA specific PNA FIT-probes neither stained noninfected cells nor cells infected by a control virus. The joint use of differently colored PNA FIT-probes in this feasibility study revealed significant differences in the expression pattern of influenza H1N1 mRNAs coding for NA or M1. These experiments provide evidence for the usefulness of PNA FIT-probes in investigations on the temporal and spatial progression of mRNA synthesis in living cells for two mRNA species.