DNA Nanotechnology as a Tool to Study Membrane-Binding Events: The Role of RGD Peptide Spatial Organization on αvβ3 Integrin Binding

Abstract

Receptor-mediated recognition, interaction and entry into cells are fundamental mechanisms in cell biology notably for the control of cell fate and behavior [1]. However, the role of specific spatial arrangements of biomolecules in the recognition and binding by receptors, as well as the kinetic parameters of these interactions for many biological mechanisms is still not yet fully understood. For example, while the RGD (arginineglycine- aspartate) peptide is a highly conserved motifs present in numerous proteins of the extra cellular matrices (ECM) involved in cell adhesion and migration its exact mechanism of interaction with cell receptors is still not fully elucidated. RGD is the principal integrinbinding domain present within ECM proteins such as vitronectin, fibronectin and fibrinogen [2]. To date, extensive efforts have been made in using RGD nanopatterns to investigate the influence of RGD organization adhesion of various cells and the relevant proliferation, migration, and differentiation behaviors [3]. However, the effect of the nanoscale organization of this peptide on the interaction with cell membrane receptors is scarce. This limited understanding is due to the lack of techniques that allow for nanoscale control of biomolecules organization and precise stoichiometry control. In 2014, Shaw and coworkers developed DNA origami nanostructures modified with ligands at well-defined positions to study the role of ligand nanoscale organization in membrane receptor-mediated signaling. They found that nanoscale spacing of ephrin-A5 regulates the invasiveness of breast cancer cells and demonstrated the usefulness of DNA origami to study this type of receptor/ligand interactions [4]. In a follow-up study Shaw et. al [5] (Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies) used DNA origami as a platform to understand the distance in which bivalent antigen could bind to a receptor. They found that the reach necessary for bivalent antigen binding for human IgG ranges from 3nm to 17 nm with strong binding affinity at 16 nm. More recently, Veneziano et. al used DNA origami nanoparticles to study the influence of number, spacing and nanoscale presentation of antigens on activation of B-cells. In their research it was found that B-cell signaling is maximized with five highly spaced antigens, and the activation increases as antigen spacing increases up to 30nm [6]. Hence, DNA origami appears a promising tool to study the impact of the nanoscale organization of biomolecules upon binding interactions with receptors [6]–[8]. Indeed, DNA is a highly programmable biomaterial with great structural predictability that can be used to build high-fidelity assemblies [9]–[11]. Moreover, DNA can be easily functionalized using classical chemistry or using single stranded DNA (ssDNA) overhangs and complementary ssDNA strands conjugated to the biomolecule of interest. Hence, molecularly precise nanoscale patterns of biomolecules can be displayed on a DNA origami structure making it realistic to study integrin-peptide interactions at the nanoscale level [4], [12]. DNA origami-RGD peptide systems represent potential for helping elucidate the role of RGD and its organization in membrane binding interactions, more specifically αvβ3 integrin binding. Using multifunctional DNA origami nanoparticles, we explored spacing, and 1D vs 2D organization of RGD peptide to bind the αvβ3 integrin at the protein level. DNA origami-RGD peptide systems not only represent potential for helping elucidate the role of RGD and its organization in membrane binding interactions but also to assemble the new generation of nanocarriers for drug delivery and effective scaffold for cell-tissue engineering.