Abstract
This report describes a novel approach to the detection of acetylcholine using DNA aptamers. Aptamers were developed by eight rounds of acetylcholine affinity column chromatography and polymerase chain reaction (PCR) amplification. Sequences from rounds 5 and 8 were screened by colorimetric enzyme-based microtiter plate assays and found to bind acetylcholine and related compounds, but not unrelated compounds. One of the highest affinity aptamers, designated ACh 6R, was further tested in aptamer-peroxidase and aptamer-fluorescence staining protocols. Using Neuro-2a murine neuroblastoma cells induced to differentiate in the presence of 1 μM all-trans-retinoic acid for 5–7 d, ACh 6R detected cholinergic cells by both the peroxidase and fluorescence methods. Unrelated DNA aptamers did not stain the cells using either method. Fixation with cold 2% paraformaldehyde was compared to cold alkaline allyl alcohol plus glutaraldehyde for immobilization of acetylcholine in situ and appeared to enable detection of greater numbers of cholinergic cells, although differences in levels of differentiation may have been a factor as well. Acetylcholine generally appeared to be distributed throughout the differentiated Neuro-2a cell bodies. However, in some cells, punctate staining along neurite outgrowths and near the termini of cellular processes suggested detection of acetylcholine in discrete vesicles.
Similar content being viewed by others
References
Bruno, J. G. In vitro selection of DNA to chloroaromatics using magnetic microbead-based affinity separation and fluorescence detection. Biochem. Biophys. Res. Comm. 234: 117–120; 1997a.
Bruno, J. G. A colorimetric inhibition study of single-stranded DNA decamer sequence interactions with dinitrotoluene. Biochem. Biophys. Res. Comm. 236: 344–346; 1997b.
Bruno, J. G., Kiel, J. L. Use of magnetic beads in selection and detection of biotoxin aptamers by ECL and enzymatic methods. BioTechniques. 32:178–183; 2002.
Bruno, J. G., Carrillo, M. P., Phillips, T., Vail, N. K., Hanson, D. Competitive FRET-aptamer-based detection of methylphosphonic acid: a common nerve agent metabolite. J. Fluorescence. In Press; 2008.
Coleman, B. A., Taylor, P. Regulation of acetylcholinesterase expression during neuronal differentiation. J. Biol. Chem. 271: 4410–4416; 1996.
Crameri, A., Stemmer, W. P. C. 1020-Fold aptamer library amplification without gel purification, Nucleic Acids Res. 21: 4410; 1993.
Curtin, B. F., Pal, N., Gordon, R. K., Namiar, M. P. Forskolin, an inducer of cAMP, up-regulates acetylcholinesterase expression and protects against organophosphate exposure in Neuro 2A cells. Molec. Cell. Biochem. 290: 23–32; 2006.
Ellington, A. D, Szostak, J W. In vitro selection of RNA molecules that bind specific ligands. Nature. 346:818–822; 1990.
Geffard, M., McRae-Degueurce, A., Souan, M. L. Immunocytochemical detection of acetylcholine in the rat central nervous system. Science. 229: 77–79; 1985a.
Geffard, M., Vieillemaringe, J., Heinrich-Rock, A. M., Duris, P. Anti-acetylcholine antibodies and first immunocytochemical application in insect brain. Neurosci. Lett. 57: 1–6; 1985b.
Hofacker, I. L. Vienna RNA secondary structure server. Nucleic Acids Res. 31: 3429–3431; 2003.
Houser, C. R. Cholinergic synapses in the central nervous system: studies of the immunocytochemical localization of choline acetyltransferase. J. Electron. Microsc. Tech. 15: 2–19; 1990.
Ikanovic. M., Rudzinski, W. E., Bruno, J. G., Allman, A., Carrillo, M. P., Dwarakanath S., Bhahdigadi, S., Rao, P., Kiel, J. L., Andrews, C. J. Fluorescence assay based on aptamer-quantum dot binding to Bacillus thuringiensis spores. J. Fluorescence. 17:193–199; 2007.
Jayasena, S. D. Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin. Chem. 45: 1628–1650; 1999.
Jenison, R. D., Gill, S. C., Pardi, A., Polisky, B. High-resolution molecular discrimination by RNA. Science 263:1425–1429; 1994.
Jones-Villeneuve, E. M. V., McBurney, M. W., Rogers, K. A., Kalnins, V. I. Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells. J. Cell Biol. 94: 253–262; 1982.
Kassil, G. N., Sokolinskaya, R. A. Mechanism of the phenomenon of fixation of acetylcholine by human blood in vitro. Bull. Exp. Biol. Med 57: 421–425; 1964.
Malik, M. A., Greenwood, C. E., Blusztajn, J. K., Berse, B. Cholinergic differentiation triggered by blocking cell proliferation and treatment with all-trans-retinoic acid. Brain Res. 874: 178–185; 2000.
Manabe, T., Tatsumi, K., Inoue, H., Matsuyoshi, H., Makinodan, M., Yokoyama, S., Wananka, A. L3/Lhx8 is involved in the determination of cholinergic or GABAergic cell fate. J. Neurochem. 94: 723–730; 2005.
Mann, D., Reinemann C., Stoltenburg, R., Strehlitz, B. In vitro selection of DNA aptamers binding ethanolamine. Biochem. Biophys. Res. Comm. 338: 1928–34; 2005.
McRae-Degueurce, A., Geffard, M. One perfusion mixture for immunocytochemical detection of noradrenaline, dopamine, serotonin and acetylcholine in the same rat brain. Brain Res. 376: 217–210; 1986.
Misawa H, Takahashi R, Deguchi T. Calcium-independent release of acetylcholine from stable cell lines expressing mouse choline acetyltransferase cDNA. J. Neurochem. 62: 465–470; 1994.
Riboni, L., Prinetti, A., Bassi, R., Caminiti, A., Tettamanti, G. A mediator role of ceramide in the regulation of neuroblastoma Neuro2a cell differentiation. J. Biol. Chem. 270: 26868–26875; 1995.
SantaLucia, J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. USA. 95: 1460–1465; 1998.
Shahed, A. R., Werchan, P. M., Stavinoha, W. B. Differences in acetylcholine but not choline in brain tissue fixed by freeze fixation or microwave heating. Meth. Find. Exp. Clin. Pharmacol. 18: 349–351, 1996.
Smith, J. E., Reese, T. S. Use of aldehyde fixatives to determine the rate of synaptic transmitter release. J. Exp. Biol. 89: 19–29; 1980.
Stanlis, K. K. H., McIntosh, J. R. Single-strand DNA aptamers as probes for protein localization in cells. J. Histochem. Cytochem. 51: 797–808; 2003.
Tsuji, S. Electron-microscope cytochemistry of acetylcholine-like cation by means of low-temperature “ionic fixation”. Histochem. Cell Biol. 81: 453–455; 1984.
Acknowledgment
Work was funded by U.S. Army contract number W911SR-06-C-0059.
Author information
Authors and Affiliations
Corresponding author
Additional information
Editor: J. Denry Sato
Rights and permissions
About this article
Cite this article
Bruno, J.G., Carrillo, M.P., Phillips, T. et al. Development of DNA aptamers for cytochemical detection of acetylcholine. In Vitro Cell.Dev.Biol.-Animal 44, 63–72 (2008). https://doi.org/10.1007/s11626-008-9086-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11626-008-9086-0