NSTI Nanotech 2009

Single molecule aptamer-target interactions on self-assembled monolayer platforms

X. Zhang, V.K. Yadavalli
Virginia Commonwealth University, VA

Keywords: biosensors, molecular recogniion


Molecular recognition between specific receptors and ligands is a key step in many biological processes and forms the basis for diverse biosensor systems. Such interactions include enzyme-substrate, antigen-antibody and oligonucleotide-target binding. Highly specific interactions between oligonucleotides and proteins are ubiquitous in nature. The discovery that nucleic acids are capable of recognizing and modulating the activity of proteins in biological systems has led to functional nucleic acids such as ribozymes, riboswitches and short synthetic oligonucleotide (DNA/RNA) sequences known as aptamers, being harnessed for a new generation of applications. In particular, biodiagnostics and biosensors are being developed to directly exploit the exquisite molecular recognition abilities of aptamers. Therapeutic aptamers are becoming a general modality for use against pathogenic proteins. As aptamers emerge as extremely valuable tools in the development of therapies and diagnostic devices, understanding the modes of interaction of aptamers and their targets is of vital importance in the rational design of drugs and devices. How an aptamer binds to its target in a physiological environment still remains unresolved. Force spectroscopy using an atomic force microscope (AFM) is a valuable tool for studying such interactions and molecular biorecognition at a single molecule level. The common practice is to apply a small concentration of protein to a surface and to use statistical techniques to infer the expected characteristics of the interaction between single molecule pairs. Whether the surface-bound proteins are indeed single and isolated remains unclear and often undesirable non-specific protein/surface interactions obscures informative features of the interaction. In this study, mixed self-assembled monolayers (SAMs) consisting of N-hydroxysuccinimide (NHS) and oligoethylene glycol (OEG)-terminated thiols on ultraflat gold surfaces were used to covalently immobilize proteins via lysine residues. By the optimization of attachment sites via lysine-NHS linkages on a protein-resistant layer of the OEG SAM, it is possible to isolate single proteins for study in a controlled fashion. Single protein distribution on the surface is clearly demonstrated by AFM imaging. OEG also significantly reduces non-specific tip-surface interactions between the AFM cantilever and surface. The utility of this self-assembled monolayer platform is demonstrated in studies of the interactions of highly specific aptamers (RNA and DNA) with target proteins. The specific interaction profiles and rupture forces for aptamer-protein binding will be demonstrated. It is envisioned that this technology will enable insight into the fundamental biophysical interactions between aptamers and their target proteins. This would greatly enhance the ability to engineer and deliver the next generation of “smart” drugs, arrays and diagnostic tools.
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