Biophysical characterization of VEGF–aHt DNA aptamer interactions

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Abstract

The binding of the well-studied DNA aptamer aHt (5′-ATACCAGTCTATTCAATTGGGCCCGTCCGTAT GGTGGGTGTGCTGGCCAG-3′), which has been demonstrated to recognize human vascular endothelial growth factor (VEGF165) to recombinant VEGF was characterized using fluorescence anisotropy, isothermal titration calorimetry and analytical ultracentrifugation. The negatively-charged DNA aptamer is selective for VEGF and does not recognize positively-charged hen egg lysozyme, or bovine serum albumin. In contrast to the VEGF association of the previously-described aV DNA aptamer, where the binding is enthalpically driven and sequence-specific, the binding of the aHt aptamer to VEGF is entropically-driven and not abolished by scrambling of the sequence.

Introduction

Aptamers are DNA or RNA oligonucleotides selected from large libraries for binding to preselected targets, which can be proteins, whole cells, or any of a variety of organic and inorganic molecules [1], [2], [3], [4]. Aptamers commonly are selected using methods related to Systematic Evolution of Ligands by EXponential enrichment (SELEX), an in vitro iterative binding/PCR selection process that allows the isolation of specific DNA or RNA molecules from a large pool of random oligonucleotides (∼1015) [5], [6].

Vascular endothelial growth factor (VEGF) has been found to be secreted by several tumor cell types and promotes tumor growth through vasculogenesis and angiogenesis [7], [8]. VEGF also is over-expressed in diabetic retinopathy and macular degeneration [9], and plays an important role in rheumatoid arthritis [10], [11]. Bevacizumab (Avastin) became the first anti-VEGF drug approved by the FDA in 2004 for the treatment of colorectal tumors [12]. Also in 2004, pegaptanib (Macugen), an RNA aptamer targeting the heparin binding domain of VEGF, became the first FDA-approved aptamer when it was cleared for the treatment of age-related macular degeneration [13], [14]. VEGF exists in four isoforms of 121, 165, 189 and 206 amino acids, of which VEGF165 is the most common form [10].

We have previously characterized the biophysical interactions between VEGF165 and the DNA aptamer aV described by Janjic and Gold [15]. Like pegaptanib, the aV DNA aptamer binds to the heparin binding domain of VEGF165 dimer with 1:1 stoichiometry. We found aV–VEGF binding to be enthalpically driven, with an unfavorable entropy change [16], a pattern also seen in the recognition of proteins by antibodies [17], [18]. In 2008, Hasegawa et al. reported another DNA aptamer (aHt) which also binds to the heparin binding domain of VEGF165 with an equilibrium dissociation constant, Kd, of 480 nM (as determined by a dot blot in which the aptamer was added to a membrane decorated with VEGF165 or with VEGF121, which lacks the heparin-binding domain) [19]. The same aHt aptamer was later used by Hasegawa et al. to construct a homodimeric aHt aptamer with 25-fold improved affinity (Kd = 17 nM) for VEGF [20]. Recently, Nonaka et al. produced a heterodimer with 1000-fold better affinity for VEGF (Kd = 470 pM) [21] by linking the aHt aptamer to another DNA aptamer recognizing the receptor binding domain of VEGF165. In this work, we have characterized the interaction of VEGF with the aHt DNA aptamer using fluorescence anisotropy, isothermal titration calorimetry (ITC) and analytical ultracentrifugation (AUC). Also, to test the sequence/structure-specificity of the binding of aHt aptamer to VEGF, we investigated the binding of two scrambled derivatives of aHt to VEGF by fluorescence anisotropy.

Section snippets

Materials

The sequences of the aHt, aHt-scr1 and aHt-scr2 aptamers were 5′-ATACCAGTCTATTCAATTGGGCCCGTCCGTATGGTGGGTGTGCTGGCCAG-3′, 5′-CCGCGGTTGGTAGGACAGGTGGAGTGTCTAGTCCAATTTGGCACTCTTCC-3′ and 5′-AAAAACCCCCCTGTGTGTGTGTGTGTGAAGACCCCCCTGTGTGTGTGTGG-3′ respectively. 5′-6-FAM labeled (aHt, aHt-scr1 and aHt-scr2) and 5′-Alexa488 labeled (aHt, for AUC experiments) aptamers were purchased from Integrated DNA Technologies, Coralville, IA, USA. The dye was attached during the solid-phase synthesis of the

Specificity of aHt–VEGF binding

The specificity of the DNA aptamer aHt (Fig. 1) for VEGF over hen egg lysozyme (HEL) and bovine serum albumin (BSA) was analyzed by monitoring the change in fluorescence anisotropy upon titration of 50 nM FAM-labeled aHt aptamer in TBSE with increasing concentrations of each protein. Positively charged HEL, known to bind non-specifically to negatively charged nucleotides [25], was used as a control along with BSA as a more neutral negative control.

The Kd for VEGF binding by the aHt aptamer was

Author contributions

Indhu Kanakaraj designed and performed experiments, interpreted data and wrote the manuscript; Wen-Hsiang Chen participated in interpreting data and writing the paper; Mohan Poongavanam performed experiments; Sagar Dhamane performed experiments; Loren J. Stagg performed experiments, and participated in interpreting data and writing the manuscript; John E. Ladbury participated in interpreting data and writing the manuscript; Katerina Kourentzi participated in interpreting data and writing the

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This research was funded in part by grants to RCW from the Welch foundation (Grant E-1264), NASA and NSF.

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