Screening Aptamers as Targeting Agents to deliver Payloads.
Aptamers are small, synthetic, single-stranded nucleic acids
that fold into unique secondary structures. Unlike peptides and monoclonal antibodies, they are essentially non-immunogenic even when administered in excess amounts.
The publications below explain how biotinylated aptamers combined with Streptavidin-conjugated saporin (Streptavidin-ZAP) can be used to screen aptamers for their ability to internalize into a specific cell type.
Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers. Hernandez, LI, Flenker, KS, Hernandez, FJ, Klingelhutz, AJ, McNamara, JO, 2nd, & Giangrande, PH. (2013). Pharmaceuticals (Basel), 6 (3):295-319. 2013/07/31. 3722562
Streptavidin-ZAP (Cat. #IT-27)
FGF-SAP (Cat. #IT-38)
Objective: isolate aptamers that internalize upon binding to their cognate receptor on the cell surface.
Summary: Among the methods used to characterize aptamers that internalize is a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Biotin-labeled A9g was conjugated to streptavidin-modified saporin (streptavidin-ZAP). First, it was verified that conjugation of biotinylated aptamer to streptavidin-ZAP (A9g-SAP) did not affect the inhibitory effect of the aptamer. Next, the effect was examined of A9g-SAP on PC3(PSMA+) and PC3(PSMA-) cells. Cells were treated with varying amounts of aptamer-saporin conjugate for 72 h at 37°C and then an assay was performed to determine potential cytotoxicity of the conjugate. Results confirm that A9g is internalized preferentially into target cells and that A9g is efficiently accessing the cytoplasm of target cells possibly through a mechanism of endosomal escape, resulting in inhibition of protein synthesis and ultimate cell-death. FGF-SAP was used as a control.
Method for Confirming Cytoplasmic Delivery of RNA Aptamers.
Dickey DD, Thomas GS, Dassie JP, Giangrande PH.
Methods Mol Biol 1364:209-217, 2016.
Single-stranded RNA aptamers have the potential to be utilized as therapeutics in numerous diseases, and can be used for the delivery of RNA interference modulators. One roadblock encountered has been the lack of a method to confirm delivery of a payload by the aptamers to the cytoplasm of cells. In this work the authors describe a protocol involving combining biotinylated aptamers and Streptavidin-ZAP at a 4:1 molar ratio, then testing the conjugates in an in vitro cytotoxicity assay. FGF-SAP was used as a control.