Department of Biology, Humboldt University Berlin, Invalidenstr. 42, D-10115 Berlin, Germany.
Bioconjug Chem. 2012 Oct 17;23(10):2051-60. doi: 10.1021/bc300249f. Epub 2012 Sep 14.
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 °C) as well as real-time PCR measurements (at 60 °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.
荧光杂交探针可用于 RNA 成像，能提供有关特定 RNA 分子在活细胞中的合成和运输是如何协调的信息。在这项研究中，我们探索了肽核酸（PNA）为基础的 FIT 探针，用于同时对甲型 H1N1 流感病毒复制周期中表达的两种不同病毒 mRNA 分子进行成像。PNA FIT 探针是非核苷酸的、无结构的探针，包含一个单一的不对称氰基染料，作为荧光碱基替代物。荧光团作为局部嵌入探针，通过增强荧光来报告靶 DNA/RNA 的杂交。虽然多色杂交探针有望促进 RNA 表达的分析，但以前没有关于两种不同病毒 mRNA 靶标双色成像的报道。在这项工作中，我们开发了一组两种不同颜色的 PNA FIT 探针，允许对编码神经氨酸酶（NA）和基质蛋白 1（M1）的 mRNA 进行光谱分辨成像；这些蛋白在流感病毒复制过程中执行不同的功能。这些探针的特点是适用杂交温度范围广泛。相同的探针序列可实现活细胞 RNA 成像（在 37°C）以及实时 PCR 测量（在 60°C 退火温度）。这通过定量（qPCR）和定性（成像）手段促进了对 RNA 表达的全面分析。共焦激光扫描显微镜显示，针对病毒 RNA 的 PNA FIT 探针既不会染色未感染的细胞，也不会染色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 的流感 H1N1 mRNA 表达模式的显著差异。这些实验为 PNA FIT 探针在研究活细胞中两种 mRNA 物种的 mRNA 合成的时空进展中的有用性提供了证据。
