Abstract
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.
Highlights
Since the discovery of RNA interference (RNAi), substantial efforts have been directed towards harnessing the sequence-specific silencing potential of small interferingRNAs for therapeutic applications
We have previously described the ability of A9g to target prostate specific membrane antigen (PSMA)
We describe both qualitative and quantitative assays for characterizing the cell-type specific internalization ability of RNA aptamers for cell-targeted therapeutic applications
Summary
Since the discovery of RNA interference (RNAi), substantial efforts have been directed towards harnessing the sequence-specific silencing potential of small interfering (si)RNAs for therapeutic applications. One such approach, based on synthetic RNA ligands (aptamers), has been employed by us and others, to facilitate delivery of siRNAs to the cytoplasm of target cells both in vitro and in vivo [4,5,6,7,8,9,10,11,12] For this approach, aptamers that bind cell-specific receptors are conjugated with partially (one strand only) or fully (both strands) chemically-modified siRNAs in chimeric molecules. The cell-internalization SELEX protocol has been useful at identifying several aptamers for siRNA-delivery [16,17], this approach does not necessarily select for aptamers that effectively escape endosomes and are released into the cytoplasm of target cells Modifications to this approach (e.g., inclusion of fractionation steps to separate endosome-bound from cytoplasmic aptamer sequences) that allow for the selective amplification of cytoplasmic-specific aptamers may enable the effective isolation of cytoplasmic-targeting RNA sequences. These methodologies promise to facilitate the development and characterization of cell-internalizing aptamers for delivery of therapeutics (e.g., siRNAs) into their target cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
