title: Capillary electrophoretic development of aptamers for a glycosylated VEGF peptide fragment.
abstract: The emergence of functional genomics and proteomics has added to the growing need for improved analysis methods that can detect and distinguish between protein variants resulting from allelic variation, mutation, or post-translational modification. Aptamers, single-stranded DNA or RNA molecules that fold into three-dimensional structures conducive to binding targets, have become an attractive alternative to antibodies for this type of analysis. Although aptamers have been developed for a wide range of target species, very few sequences have been identified that bind selectively to proteins with specific post-translational modifications. Using capillary electrophoresis-based selection, we have developed DNA aptamer sequences that selectively bind an N-glycosylated peptide fragment of vascular endothelial growth factor (VEGF). The selection method incorporates alternating positive- and counter-selection steps in free solution in order to obtain aptamers with both high affinity toward the glycosylated target and high selectivity versus a non-glycosylated variant. Affinity capillary electrophoresis and surface plasmon resonance binding assays indicate these sequences have low-µM dissociation constants and preferentially bind the glycosylated peptide with as much as 50-fold specificity. Such aptamers could serve as tools for rapid and simple monitoring of disease-linked functional changes in proteins, with potential applications in drug screening and disease diagnosis.
authors.affiliation: Department of Chemistry and Biochemistry, Santa Clara University, Santa Clara, CA, USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Rose
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hayes
authors.forename: Gregory R
authors.initials: GR
authors.lastname: Stettler
authors.forename: Scott F
authors.initials: SF
authors.lastname: Hickey
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Axelrod
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Giustini
authors.forename: Steven W
authors.initials: SW
authors.lastname: Suljak
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001665:Binding Sites; D019075:Electrophoresis, Capillary; D006031:Glycosylation; D010446:Peptide Fragments / Q000032:analysis*; D020349:Surface Plasmon Resonance; D042442:Vascular Endothelial Growth Factors / Q000032:analysis* / Q000737:chemistry
chemical_list: D052157:Aptamers, Nucleotide; D010446:Peptide Fragments; D042442:Vascular Endothelial Growth Factors
references.citation: Chem Commun (Camb). 2006 Aug 14;(30):3229-31
references.pmid: 17028752
references.citation: Anal Chem. 2004 Sep 15;76(18):5387-92
references.pmid: 15362896
references.citation: J Am Chem Soc. 2009 May 13;131(18):6330-1
references.pmid: 19385619
references.citation: Biophys J. 2009 Sep 16;97(6):1804-7
references.pmid: 19751687
references.citation: Science. 1990 Aug 3;249(4968):505-10
references.pmid: 2200121
references.citation: Bioorg Med Chem. 1997 Jun;5(6):1115-22
references.pmid: 9222505
references.citation: Anal Chem. 2005 Oct 1;77(19):6107-12
references.pmid: 16194066
references.citation: Anal Chem. 1998 Nov 1;70(21):4540-5
references.pmid: 9823713
references.citation: Molecules. 2010 Mar 02;15(3):1127-40
references.pmid: 20335968
references.citation: Biochim Biophys Acta. 1999 Dec 6;1473(1):4-8
references.pmid: 10580125
references.citation: Nature. 2007 Jun 7;447(7145):741-4
references.pmid: 17554312
references.citation: Anal Chim Acta. 2006 Mar 30;564(1):91-6
references.pmid: 17723366
references.citation: Nature. 1990 Aug 30;346(6287):818-22
references.pmid: 1697402
references.citation: Science. 1989 Dec 8;246(4935):1306-9
references.pmid: 2479986
references.citation: Langmuir. 2009 May 5;25(9):5054-60
references.pmid: 19253965
references.citation: Clin Chem. 1999 Sep;45(9):1628-50
references.pmid: 10471678
references.citation: Anal Chem. 2010 Mar 1;82(5):2042-7
references.pmid: 20136122
references.citation: J Am Chem Soc. 2004 Jan 14;126(1):20-1
references.pmid: 14709039
references.citation: J Am Chem Soc. 2005 Jul 6;127(26):9382-3
references.pmid: 15984861
references.citation: J Am Chem Soc. 2008 Sep 24;130(38):12636-8
references.pmid: 18763762