Development of DNA aptamers for cytochemical detection of acetylcholine

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-b...

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Bibliographic Details
Published in:In vitro cellular & developmental biology. Animal 2008-03, Vol.44 (3), p.63-72
Main Authors: Bruno, John G, Carrillo, Maria P, Phillips, Taylor, King, Blythe
Format: Article
Language:English
Subjects:
Acetylcholine
Acetylcholine - analysis
Animal Genetics and Genomics
Animals
Aptamer
Aptamers, Nucleotide
Biomedical and Life Sciences
Biotechnology
Cell Biology
Cell Compartmentation
Cell Culture
Cell Line
Cell lines
Cellular differentiation
Cholinergic receptors
Cholinergics
Chromatography, Affinity
Cytochemistry
Developmental Biology
DNA
Fluorescence
Histochemistry
Life Sciences
Mice
Molecules
Neuroblastoma
Neuroglia
Neurons
Neurotransmitter
Nucleic Acid Conformation
Nucleotide aptamers
SELEX
SELEX Aptamer Technique
Stem Cells
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Summary: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.
ISSN:1071-2690
1543-706X
DOI:10.1007/s11626-008-9086-0