Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers.
Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers.
复制标题
DOI：
10.3390/ph6030295
复制
发表时间：
2013-03-14
期刊：
影响因子：
--
通讯作者：
Giangrande PH
中科院分区：
文献类型：
--
作者：
Hernandez LI;Flenker KS;Hernandez FJ;Klingelhutz AJ;McNamara JO 2nd;Giangrande PH
最近的小干扰RNA（siRNA）的临床试验强调了对强大的递送技术的需求，这些技术将有助于将这些治疗药物成功应用于人类。可以说，通过与细胞特异性配体缀合的细胞靶向为该问题提供了可行的解决方案。合成的RNA配体（适体）代表了一类新兴的药物，具有靶向治疗应用的巨大潜力。为了用适体靶向递送siRNA，适体-siRNA缀合物必须被细胞吸收并到达细胞质。为此，我们已经开发了基于细胞的选择方法来分离适体，所述适体在与细胞表面上的其同源受体结合后内化。在这里，我们描述了监测适体的细胞摄取的方法。其中包括：（1）抗体扩增显微术，（2）基于微孔板的荧光测定，（3）定量和超灵敏内化方法（“QUSIM”）和（4）使用基于核糖体失活蛋白（RNA-RIP）测定监测细胞质递送的方法。总的来说，这些方法提供了一套工具，可以加快适体配体的开发，以在体内靶向和递送治疗性siRNA。
Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method (“QUSIM”) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.