Proteins aptamers

To study the reproducibility and the reusability of the sensor, successive washing steps were performed with solutions of 8 M Urea and 0.2 M Ca EDTA followed by re-incubation with 10 nM thrombin. Urea was chosen to destabilize the protein aptamer complex and Ca /EDTA was chosen to disrupt the G-quadruplex. Spectra recorded after washing exhibit the low intensity unbound signal and signal increases repeatedly after incubation with 10 nM thrombin - these two states can be... 

Antibody fragment libraries from immunized and nonimmunized sources can be used in phage display and peptide libraries are commercially available. An alternative technique is to design protein aptamers that consist of a stable protein scaffold on which random peptides are displayed. An example of protein aptamers are affibodies, which present a library of 13 randomized amino acids on the Z domain of Staphylococcus aureus protein A. Crystal structure studies indicate similarity in the binding of an affibody to its target to protein-antibody interactions. However, affibodies have a dissociation constant of approximately 1 /.tM compared to antibody-antigen complexes ofl nM orless(H3, Rl, Wl). 

Li, Y., Lee, H. J., Corn, R. M. (2006). Fabrication and characterization of RNA aptamer microarrays for the study of protein-aptamer interactions with SPR imaging. Nucleic Acids Res 34, 6416-6424. 

Cox, J. C., Ellington, A. D. (2001). Automated selection of anti-protein aptamers. Bioorg Med Chem 9, 2525-2531. 

DETECTION OF PROTEIN-APTAMER INTERACTIONS BY MEANS OF ELECTROCHEMICAL INDICATORS AND TRANSVERSE SHEAR MODE METHOD... 

In this chapter the focus is on the use of electrochemical indicators and of the TSM method for the study of protein-aptamer interactions. Aptamers sensitive to thrombin are used for the fabrication of biosensors. Attention is given to methods of immobilization of aptamers to a solid support and how this affects the interaction of thrombin with the aptamer. The effects of aptamer structure, of the presence of the thrombin inhibitor heparin, and of the ions and pH on the binding properties of aptamer are presented. The advantages of electrochemical indicators and the TSM method for the detection of thrombin-aptamer interactions are demonstrated. 

In its simplest, QCM, format, protein-aptamer interactions were analyzed by Liss et al. (2002). They compared the interaction of IgE with DNA aptamer as well as with anti-IgE antibodies. Although the detection limit was similar in the two cases, the advantage of the aptasensor was its possibility of surface regeneration, which was impossible for an antigen-based biosensor. However, recently it has been shown that immobilization of anti-IgE on the dendrimer surface also allows us to regenerate an immunosensor (Svobodova et al., 2006). The QCM method was recently compared with the electrochemical biosensor assay of thrombin detection (Hianik et al., 2005, 2007). It has been shown that the sensitivity of thrombin detection was similar for the two methods. Mascini and co-workers showed that similar results in sensitivity and selectivity in the detection of Tat peptide with RNA aptamer can be obtained by the QCM and SPR methods (Tombelli et al., 2005b). 

Thus, acoustic sensors represent a rather useful tool for the study of protein-aptamer interactions without additional labeling (Bini et al., 2007). Using an acoustic sensor in a TSM format, it is also possible to smdy subtle... 

Berezovski. M., Nutiu, R., Li, Y., Krylov, S. N. (2003). Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures. Anal Chem 75, 1382-1386. 

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