title: High efficiency acetylcholinesterase immobilization on DNA aptamer modified surfaces.
abstract: We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE). One selected aptamer sequence (R15/19) has a high affinity towards the enzyme (Kd=157±42 pM). Characterization of the aptamer showed its binding is not affected by low ionic strength (~20 mM), however significant reduction in affinity occurred at high ionic strength (~1.2 M). In addition, this aptamer does not inhibit the catalytic activity of AChE that we exploit through immobilization of the DNA on a streptavidin-coated surface. Subsequent immobilization of AChE by the aptamer results in a 4-fold higher catalytic activity when compared to adsorption directly on to plastic.
authors.affiliation: Department of Chemistry, Imperial College, London SW7 2AZ, UK.
authors.forename: Orada
authors.initials: O
authors.lastname: Chumphukam
authors.affiliation: Department of Chemistry, Imperial College, London SW7 2AZ, UK.
authors.forename: Thao T
authors.initials: TT
authors.lastname: Le
authors.affiliation: Department of Chemistry, Imperial College, London SW7 2AZ, UK. t.cass@imperial.ac.uk.
authors.forename: Anthony E G
authors.initials: AE
authors.lastname: Cass
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry*; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D004593:Electrophorus; D004800:Enzymes, Immobilized / Q000737:chemistry*; D029941:Fish Proteins / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D011485:Protein Binding; D052156:SELEX Aptamer Technique; D019809:Streptavidin / Q000737:chemistry
chemical_list: D052157:Aptamers, Nucleotide; D004800:Enzymes, Immobilized; D029941:Fish Proteins; D019809:Streptavidin; D000110:Acetylcholinesterase
references.citation: Nat Rev Genet. 2008 Apr;9(4):291-302
references.pmid: 18347592
references.citation: J Biotechnol. 2010 Feb 15;145(4):317-22
references.pmid: 20026208
references.citation: J Mol Biol. 2000 Sep 8;302(1):79-91
references.pmid: 10964562
references.citation: Enzyme Microb Technol. 2011 Feb 8;48(2):107-22
references.pmid: 22112819
references.citation: Hum Mol Genet. 2003 Apr 1;12 Spec No 1:R1-8
references.pmid: 12668591
references.citation: Nat Biotechnol. 1996 Aug;14(8):1021-5
references.pmid: 9631044
references.citation: Nature. 1992 Feb 6;355(6360):564-6
references.pmid: 1741036
references.citation: J Immunol Methods. 1992 Jun 24;150(1-2):77-90
references.pmid: 1613260
references.citation: Methods. 2005 Sep;37(1):4-15
references.pmid: 16199170
references.citation: Biochem Pharmacol. 1961 Jul;7:88-95
references.pmid: 13726518
references.citation: Anal Biochem. 2002 Oct 1;309(1):85-95
references.pmid: 12381366
references.citation: Protein Sci. 2001 Dec;10(12):2608-17
references.pmid: 11714929
references.citation: Nucleic Acids Res. 2002 May 15;30(10):e45
references.pmid: 12000850
references.citation: Anal Biochem. 2005 Mar 1;338(1):113-23
references.pmid: 15707941
references.citation: Biochemistry. 1980 Aug 5;19(16):3705-11
references.pmid: 7407068
references.citation: J Biol Chem. 1999 Oct 22;274(43):30370-6
references.pmid: 10521413
references.citation: Annu Rev Biochem. 2005;74:53-82
references.pmid: 15952881
references.citation: J Biochem Biophys Methods. 2001 Feb 26;47(3):221-31
references.pmid: 11245893
references.citation: Biotechnol Prog. 1992 Jul-Aug;8(4):268-74
references.pmid: 1368450
references.citation: J Am Chem Soc. 2007 Dec 5;129(48):14966-73
references.pmid: 17997555
references.citation: Protein Expr Purif. 2005 May;41(1):98-105
references.pmid: 15802226
references.citation: Methods Mol Biol. 2004;264:101-10
references.pmid: 15020783
references.citation: Annu Rev Anal Chem (Palo Alto Calif). 2009;2:241-64
references.pmid: 20636061
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
references.pmid: 12824337
references.citation: Chem Rev. 2008 Jan;108(1):109-39
references.pmid: 18095717
references.citation: PLoS One. 2010 Dec 07;5(12):e15004
references.pmid: 21165148
references.citation: J Mol Biol. 1997 Oct 10;272(5):688-98
references.pmid: 9368651
references.citation: Nat Rev Drug Discov. 2010 Jul;9(7):537-50
references.pmid: 20592747
references.citation: Proteomics. 2013 Feb;13(3-4):457-66
references.pmid: 23255229
references.citation: Nat Rev Drug Discov. 2006 Feb;5(2):123-32
references.pmid: 16518379
references.citation: Nucleic Acids Res. 2000 Jul 15;28(14):E71
references.pmid: 10908345
references.citation: Science. 1994 Mar 11;263(5152):1425-9
references.pmid: 7510417
references.citation: Phys Chem Chem Phys. 2011 Mar 28;13(12):5271-8
references.pmid: 21344100
references.citation: Mol Immunol. 1993 Sep;30(13):1165-75
references.pmid: 8413321
references.citation: RNA. 2001 Apr;7(4):632-41
references.pmid: 11345441