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.

Tracking the Emergence of High Affinity Aptamers for rhVEGF165 During Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment Using High Throughput Sequencing

“…The affinities obtained for the individual aptamers were also similar to those of measured for the oligonucleotide pools throughout the selection and were comparable to or better than previously reported DNA aptamers selected against VEGF 165 or VEGF peptide. 24,25,32 It should be noted that none of the 27 previously reported aptamer sequences for VEGF were observed any of our selected pools. This is not surprising since different primers or target preparations would be expected to significantly affect the aptamer sequences that are selected.…”

Post-translational modifications in tumor biomarkers: the next challenge for aptamers?

“…The use of synthetic peptides as targets allows the incorporation of counter-selection steps using a non-glycosylated synthetic variant, which is another possibility to direct aptamers towards the glycosylation site. This approach has been employed for the selection of a DNA aptamer that selectively bind an N-glycosylated peptide fragment of vascular endothelial growth factor (VEGF) [25]. Despite only one unit of Nacetylglucosamine is incorporated at the natural glycosylation site of VEGF, the resulting aptamers show 52-fold selectivity towards the glycan-peptide over the non-glycosylated variant.…”

Capillary electrophoresis applied to DNA: determining and harnessing sequence and structure to advance bioanalyses (2009–2014)

“…Capillary electrophoresis-SELEX methods [ 115 – 120 ] have recently been used to generate aptamers predominantly for proteins, although work with peptides [ 121 ] and small molecules [ 122 ] is reported. Innovations in the field of capillary electrophoresis-SELEX continue.…”

“…Pre-equilibrium affinity capillary electrophoresis is used when the aptamer complex does not dissociate significantly during the time frame of the separation. Pre-incubation equilibrium affinity capillary electrophoresis has been applied to proteins [ 114 , 116 , 117 , 123 , 148 ], peptides [ 121 ], and small molecules [ 117 , 149 ]. An innovative application utilizes a micro free-flow device for affinity capillary electrophoresis as a means to sample the ratio of bound and free aptamer at a wide range of concentrations.…”