|Wang, Bin -|
|Guo, Cunlan -|
|Zhang, Mengmeng -|
|Xu, Bingqian -|
Submitted to: Journal of Physical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 8, 2012
Publication Date: April 10, 2012
Citation: Wang, B., Guo, C., Zhang, M., Park, B., Xu, B. 2012. High-resolution single-molecule recognition imaging of the molecular details of ricin-aptamer interaction. Journal of Physical Chemistry. B116(17):5316-22. Interpretive Summary: Atomic Force Microscopy (AFM) has been widely used for nanotechnology to detect biospecies in vitro and in situ, because the AFM recognition imaging and force spectroscopy have the advantage to probe topography images and molecular recognition simultaneously and is able to detect biomolecules in single-molecule level. Aptamers are single-stranded oligonucleotides generated from in vitro selection and have high affinities to their targets, which can be small molecules, proteins, virus, and cells. We modified AFM tip with aptamer to detect single ricin protein and investigated binding affinity of aptamer to ricin. The affinity between aptamer and its target species is based on the structure-function relationship of these biospecies. Since aptamers are increasingly used for the biosensor research, our goal is to develop novel biosensors for detecting various food toxins based on DNA aptamers. In doing this, the AFM images and computer simulation model were used to compare aptamer with antibody for their binding affinities to ricin. We found that the aptamer showed slightly higher affinity to ricin than antibody. We also developed the methods for surface immobilizations of ricin and aptamer, and tested the affinity and specificity.
Technical Abstract: The molecular details of DNA aptamer-ricin interactions were investigated. The toxic protein ricin molecules were immobilized on Au(111) surface using N-hydroxysuccinimide (NHS) ester to specifically react with lysine residues located on the ricin B chains. A single ricin molecule was visualized in situ using the AFM tip modified with an anti-ricin aptamer. Computer simulation was conducted to illustrate the protein and aptamer structures, the single-molecule ricin images on Au(111) surface, and binding conformations of ricin-aptamer and ricin-antibody complexes. The various ricin conformations on Au(111) surface were caused by different lysine residues reacting with NHS ester. It was also observed that most of the binding sites for aptamer and antibody on the A chains of ricin molecules were not interfered by the immobilization reaction. The different locations of ricin binding sites to aptamer and antibody were distinguished by AFM recognition images as well as simulations. To determine the specific binding sites of aptamers and antibody to the ricin molecules, interaction blocking experiment was performed using aptamer solution. The recognition signals were reduced significantly 46 min after injection of aptamer solution, which means ricin binding sites to aptamer were blocked by other aptamers efficiently. These experimental results were very useful for validation of simulation results for the binding conformation.