Method for Confirming Cytoplasmic Delivery of RNA Aptamers.
Method for Confirming Cytoplasmic Delivery of RNA Aptamers.
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DOI：
10.1007/978-1-4939-3112-5_17
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发表时间：
2016
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影响因子：
--
通讯作者：
Giangrande PH
中科院分区：
文献类型：
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作者：
Dickey DD;Thomas GS;Dassie JP;Giangrande PH
RNA适体是单链RNA寡核苷酸，代表了一种强大的新兴技术，具有治疗多种疾病的潜力。最近，已经开发了靶向细胞的RNA适体，用于将RNA干扰（RNAi）调节剂（siRNA和miRNA）递送至特定的患病细胞（例如，癌细胞或HIV感染的细胞）。然而，尽管最初的报道是有希望的，但这种适体递送技术的广泛应用仍有待于开发能够验证和确认适体递送至RNAi机制所在的靶细胞的细胞质的方法。我们最近开发了一种功能测定法（RIP测定法），以确认细胞摄取和随后的RNA适体的细胞质释放，该RNA适体结合前列腺癌细胞（PSMA）上表达的细胞表面受体。为了评估细胞质递送，将适体与皂草素化学缀合，皂草素是一种核糖体失活蛋白毒素，其仅在通过细胞靶向配体（例如，适体）。在这里，我们描述了用于将适体与皂草素缀合的化学，并讨论了基于凝胶的方法来验证缀合效率。我们还详细介绍了一种体外功能测定，以确认适体保留功能后，共轭皂草素和描述的细胞测定，以测量适体介导的皂草素诱导的细胞毒性。
RNA aptamers are single-stranded RNA oligos that represent a powerful emerging technology with potential for treating numerous diseases. More recently, cell-targeted RNA aptamers have been developed for delivering RNA interference (RNAi) modulators (siRNAs and miRNAs) to specific diseased cells (e.g., cancer cells or HIV infected cells) in vitro and in vivo. However, despite initial promising reports, the broad application of this aptamer delivery technology awaits the development of methods that can verify and confirm delivery of aptamers to the cytoplasm of target cells where the RNAi machinery resides. We recently developed a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA). To assess cytoplasmic delivery, the aptamer was chemically conjugated to saporin, a ribosome inactivating protein toxin that is toxic to cells only when delivered to the cytoplasm (where it inhibits the ribosome) by a cell-targeting ligand (e.g., aptamer). Here, we describe the chemistry used to conjugate the aptamer to saporin and discuss a gel-based method to verify conjugation efficiency. We also detail an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity.