Fluorescence Correlation Spectroscopy (FCS) is a promising tool to study interactions on a single molecule level. The diffusion of fluorescent molecules in and out of the excitation volume of a confocal microscope leads to the fluorescence fluctuations that give information on the average number of fluorescent molecules present in the excitation volume and their diffusion coefficients. In this context, we complexed mRNA into lipoplexes and polyplexes and explored the association/dissociation degree of complexes by using gel electrophoresis and FCS. FCS enabled us to measure the association and dissociation degree of mRNA-based complexes both in buffer and protein-rich biological fluids such as human serum and ascitic fluid, which is a clear advantage over gel electrophoresis that was only applicable in protein-free buffer solutions. Furthermore, following the complex stability in buffer and biological fluids by FCS assisted to understand how complex characteristics, such as charge ratio and strength of mRNA binding, correlated to the transfection efficiency. We found that linear polyethyleneimine prevented efficient translation of mRNA, most likely due to a too strong mRNA binding, whereas the lipid based carrier Lipofectamine® messengerMAX did succeed in efficient release and subsequent translation of mRNA in the cytoplasm of the cells. Overall, FCS is a reliable tool for the in depth characterization of mRNA complexes and can help us to find the critical balance keeping mRNA bound in complexes in the extracellular environment and efficient intracellular mRNA release leading to protein production.

Statement of Significance

The delivery of messenger RNA (mRNA) to cells is promising to treat a variety of diseases. Therefore, the mRNA is typically packed in small lipid particles or polymer particles that help the mRNA to reach the cytoplasm of the cells. These particles should bind and carry the mRNA in the extracellular environment (e.g. blood, peritoneal fluid, …), but should release the mRNA again in the intracellular environment. In this paper, we evaluated a method (Fluorescence Correlation Spectroscopy) that allows for the in depth characterization of mRNA complexes and can help us to find the critical balance keeping mRNA bound in complexes in the extracellular environment and efficient intracellular mRNA release leading to protein production.

中文翻译：

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荧光相关光谱法发现mRNA复合物的细胞外缔合与细胞内解离之间的关键平衡

荧光相关光谱法（FCS）是研究单分子水平相互作用的有前途的工具。荧光分子在共焦显微镜激发体积内外的扩散会导致荧光波动，从而提供有关激发体积中存在的荧光分子的平均数量及其扩散系数的信息。在这种情况下，我们将mRNA复合为脂质体和多链体，并通过凝胶电泳和FCS探索了复合体的缔合/解离度。FCS使我们能够测量缓冲液和富含蛋白质的生物液（例如人血清和腹水中）中基于mRNA的复合物的缔合和解离度，这与仅适用于无蛋白缓冲液的凝胶电泳相比，具有明显的优势。此外，通过FCS在缓冲液和生物液体中的复杂稳定性，我们可以了解复杂的特征（例如电荷比和mRNA结合强度）如何与转染效率相关。我们发现线性聚乙烯亚胺阻止了mRNA的有效翻译，这很可能是由于过强的mRNA结合，而基于脂质的载体Lipofectamine®messengerMAX确实成功地在细胞质中有效释放了mRNA，并随后进行了翻译。总体而言，FCS是深入表征mRNA复合物的可靠工具，可以帮助我们找到在细胞外环境中保持复合物中的mRNA结合以及有效的细胞内mRNA释放导致蛋白质生产的关键平衡。

重要声明

将信使RNA（mRNA）传递到细胞有望治疗多种疾病。因此，mRNA通常堆积在小的脂质颗粒或聚合物颗粒中，这些颗粒或聚合物颗粒帮助mRNA到达细胞的细胞质。这些颗粒应在细胞外环境（例如血液，腹膜液等）中结合并携带mRNA，但应在细胞内环境中再次释放mRNA。在本文中，我们评估了一种方法（荧光相关光谱法），该方法可以深入表征mRNA复合物，并可以帮助我们找到在细胞外环境中保持mRNA结合在复合物中的关键平衡以及有效的细胞内mRNA释放导致蛋白质生产的方法。