The objective of this study was to analyze the effects of fluorophores on the intracellular accumulation and biological activity of small interfering RNA (siRNA) and its cholesterol conjugates. In this study, we used stem-loop real-time PCR and calibration curves to quantitate cellular siRNA accumulation. Attachment of fluorophores significantly affected both the accumulation and biological activity of siRNA conjugates. The severity of this effect depended significantly on the structure of the conjugate; fluorophores (Cy5.5 or Alexa-488) attached to siRNA, facing the side of the duplex opposite to cholesterol, enhanced the unproductive intracellular accumulation of the conjugate when delivered in carrier-free mode. Enhanced cellular accumulation of siRNA conjugates did not result in enhanced biological activity of the conjugate. Moreover, the attachment of a hydrophobic fluorophore, such as Cy5.5, to conventional siRNA also enhanced its apparent intracellular accumulation, but not its biological activity. Thus, the use of fluorescent labels for the study of the intracellular accumulation of siRNA and its conjugates formed with different molecules is possible only for a limited range of structures, and requires verification using alternative methods.

中文翻译：

---

荧光团标记影响胆固醇修饰的siRNA的细胞积累和基因沉默活性。

这项研究的目的是分析荧光团对小分子干扰RNA（siRNA）及其胆固醇结合物的细胞内积累和生物学活性的影响。在这项研究中，我们使用茎环实时PCR和校正曲线来定量细胞siRNA的积累。荧光团的附着会显着影响siRNA共轭物的积累和生物学活性。这种作用的严重程度在很大程度上取决于结合物的结构。当以无载体模式递送时，附着于siRNA的荧光团（Cy5.5或Alexa-488），面对与胆固醇相反的双链体一侧，可增强缀合物的非生产性细胞内积累。siRNA缀合物的细胞积累增强并未导致缀合物的生物学活性增强。此外，疏水性荧光团（例如Cy5.5）与常规siRNA的连接也增强了其明显的细胞内积累，但没有增强其生物学活性。因此，仅在有限的结构范围内，将荧光标记用于研究siRNA及其与不同分子形成的结合物的细胞内积累是可能的，并且需要使用替代方法进行验证。