Probing High Affinity Sequences of DNA Aptamer against VEGF165

Harleen Kaur,Lin-Yue Lanry Yung

doi:10.1371/journal.pone.0031196

Abstract

Vascular endothelial growth factor (VEGF165) is a potent angiogenic mitogen commonly overexpressed in cancerous cells. It contains two main binding domains, the receptor-binding domain (RBD) and the heparin-binding domain (HBD). This study attempted to identify the specific sequences of the VEa5 DNA aptamer that exhibit high binding affinity towards the VEGF165 protein by truncating the original VEa5 aptamer into different segments. Using surface plasmon resonance (SPR) spectroscopy for binding affinity analysis, one of the truncated aptamers showed a >200-fold increase in the binding affinity for HBD. This truncated aptamer also exhibited high specificity to HBD with negligible binding affinity for VEGF121, an isoform of VEGF lacking HBD. Exposing colorectal cancer cells to the truncated aptamer sequence further confirmed the binding affinity and specificity of the aptamer to the target VEGF165 protein. Hence, our approach of aptamer truncation can potentially be useful in identifying high affinity aptamer sequences for the biological molecules and targeting them as antagonist for cancer cell detection.

Highlights

Short single stranded nucleic acids referred to as aptamers are widely being explored as molecules of high affinity and specificity for binding a diverse array of target molecules ranging from high molecular weight proteins to small ions and nucleotides [1,2,3]
CM5 sensor chips were purchased from GE Healthcare for protein immobilization. 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and ethanolamine-HCl were purchased from Sigma-Aldrich
Based on the surface plasmon resonance (SPR) measurement, the original VEa5 aptamer exhibited a binding constant of Kd = 120 nM to the surface immobilized VEGF165 (Table 1). This value is very close to the Kd value of the VEa5 aptamer binding to the heparin-binding domain (HBD) of VEGF165 reported in the literature (Kd = 130 nM) [35]

Summary

Introduction

Short single stranded nucleic acids referred to as aptamers are widely being explored as molecules of high affinity and specificity for binding a diverse array of target molecules ranging from high molecular weight proteins to small ions and nucleotides [1,2,3]. The non-binding domain may interfere with the interaction between the aptamer and target protein by formation of complex secondary structures, and eventually prevents the binding domain to fold into the desired conformation for binding to the target [4] This may result in reduction or complete loss of the binding affinity as well as higher synthesis cost. Software algorithms have been used to deduce the binding domains by comparing different sequences as well as to predict the secondary structure [27,28] Strategies such as partial fragmentation, enzymatic footprinting, and recently microarray based binding sequence determination have been employed for probing the high affinity binding sequences [13,29,30]. We attempted to identify the high affinity binding sequences within the 66mer VEa5 by truncating its stem-loop regions and investigated the impact on the binding affinity against HBD of VEGF165 protein using surface plasmon resonance (SPR) spectroscopy. In vitro binding study with colorectal cancer cells overexpressed with VEGF protein further confirmed the high binding affinity of the truncated aptamer

Materials and Methods

Results and Discussion

Conclusions