Abstract
High-affinity aptamers can be derived de novo by using stringent conditions in SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments or can be engineered post SELEX via dimerization of selected aptamers. Using electrophoretic mobility shift assays, we studied a series of heterodimeric and homodimeric aptamers, constructed from two DNA aptamers with distinct primary sequences and secondary structures, previously isolated for VEGF-165, a homodimeric protein. We investigated four factors envisaged to impact the affinity of a dimeric aptamer to a dimeric protein: (1) length of the linker between two aptamer domains, (2) linking orientation, (3) binding-site compatibility of two component aptamers in a heterodimeric aptamer, and (4) steric acceptability of the two identical aptamers in a homodimeric aptamer. All heterodimeric aptamers for VEGF-165 were found to exhibit monomeric aptamer-like affinity and the lack of affinity enhancement was attributed to binding-site overlap by the constituent aptamers. The best homodimeric aptamer showed 2.8-fold better affinity than its monomeric unit (Kd = 13.6 ± 2.7 nM compared to 37.9 ± 14 nM), however the barrier to further affinity enhancement was ascribed to steric interference of the constituent aptamers. Our findings point to the need to consider the issues of binding-site compatibility and spatial requirement of aptamers for the development of dimeric aptamers capable of bivalent recognition. Thus, determinants highlighted herein should be assessed in future multimerization efforts.
Highlights
Multivalent interactions are ubiquitous in nature[1]
We carried out a comprehensive investigation examining the feasibility of creating high-affinity dimeric aptamers using three different DNA aptamers previously reported for human vascular endothelial growth factor 165 (VEGF-165)[24,25,26,27,28,29,30]
We selected the technique of electrophoretic mobility shift assays (EMSA) to assess aptamer binding to VEGF-165 because this technique allows for direct observation of the protein-aptamer complex[53]
Summary
Multivalent interactions are ubiquitous in nature[1]. For example, DNA binding sites for transcription factors can occur in clusters, which are bound by oligomeric transcription factors during transcriptional control[2]. Unlike the human thrombin-DNA aptamer system[38,39,40,41,42,43,44,45] that has been the subject of many previous heterodimeric aptamer engineering efforts[6,13,17,19,22,46,47,48,49,50,51,52] (see Supplementary Table S2), for aptamer/VEGF-165 systems, no high-resolution structural data are available For this reason, lessons learned from such a system can serve as generalizable guiding principles for other protein-aptamer systems
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