title: Method for Confirming Cytoplasmic Delivery of RNA Aptamers.
abstract: 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.
authors.affiliation: Department of Internal Medicine, University of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, 52242, USA.
authors.forename: David D
authors.initials: DD
authors.lastname: Dickey
authors.affiliation: Department of Internal Medicine, University of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, 52242, USA.
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Thomas
authors.affiliation: Department of Internal Medicine, University of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, 52242, USA.
authors.forename: Justin P
authors.initials: JP
authors.lastname: Dassie
authors.affiliation: Department of Internal Medicine, University of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, 52242, USA. paloma-giangrande@uiowa.edu.|Department of Radiation Oncology, University of Iowa, Iowa City, IA, 52242, USA. paloma-giangrande@uiowa.edu.
authors.forename: Paloma H
authors.initials: PH
authors.lastname: Giangrande
mesh_terms: D000954:Antigens, Surface / Q000378:metabolism; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D004337:Drug Carriers / Q000378:metabolism* / Q000633:toxicity; D043425:Glutamate Carboxypeptidase II / Q000378:metabolism; D006801:Humans; D034622:RNA Interference; D054789:Ribosome Inactivating Proteins, Type 1 / Q000378:metabolism* / Q000633:toxicity; D000076985:Saporins
chemical_list: D000954:Antigens, Surface; D052157:Aptamers, Nucleotide; D004337:Drug Carriers; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II
keywords: Aptamers; Cytoplasmic delivery; Cytotoxicity; Saporin