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Internalization of the DNA TLS‐11a aptamer in MEAR liver hepatoma cells
Author(s) -
Stecker John R,
Bruno John G,
Koke Joseph R
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.622.5
Subject(s) - internalization , aptamer , streptavidin , biotinylation , chemistry , in vitro , cytoplasm , biophysics , microbiology and biotechnology , confocal microscopy , cell culture , confocal , cell , biochemistry , biology , biotin , genetics , geometry , mathematics
We studied the binding properties of an aptamer developed for the BNL 1ME A.7R.1 (MEAR) mouse hepatoma cell line (TLS‐11a, Shangguan, D, et al., Anal Chem 80, no. 3: 721–728) by exposing MEAR cells in vitro to the biotinylated TLS‐11a in MEM for 60 minutes at 37°C. The cells were washed with MEM and then probed with streptavidin conjugated to AlexaFluor 568 (in MEM) for 30 minutes. The streptavidin‐containing MEM was replaced with normal MEM, and the cells examined immediately by 3‐dimensional laser scanning confocal microscopy. As expected, the streptavidin‐AlexaFluor probe revealed TLS‐11a binding to the surface of MEAR cells in vitro. However, 24 hours after exposure of MEAR cells in vitro to labeled TLS‐11a, a clear change in the location of the TLS‐11a was observed; from that consistent with external membrane labeling to that consistent with internalization and compartmentalization in the cytoplasm of the MEAR cells. The internalization was slower at room temperature than at 37°C, suggesting an active endocytotic mechanism. Additional work is in progress to determine the kinetics of internalization and to use organelle specific dyes to determine the location of the non‐nuclear internalized aptamer. This finding suggests a route to deliver a toxin directly to the interior of the cell. This work is supported by internal funding from OTC Biotechnologies, LLC, and NSF DBI‐0821252 to Joseph Koke.