APTAMER MICROARRAY DEVELOPMENT FOR HUMAN THROMBIN DETECTION: INTERACTION ANALYSIS IN SOLUTION AND IN SOLID PHASE

Aptamers are RNA or single stranded DNA molecules that selectively bind to molecular targets with high specificity and affinity, making them attractive alternatives to the commonly used antibodies. Besides lower production costs, added advantages over antibodies are their relative ease of isolation and modifications, tailored binding affinity and reversible denaturation, making them suitable candidates for use as detection systems. An aptamer-based microarray for human thrombin detection has been developed, exploiting two non-overlapping DNA antithrombin aptamers recognizing different exosites of the target protein. TBA1 and TBA2 have been used: the 15-mer aptamer (TBA1) binds the fibrinogen binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. This distinct recognition pattern allows their use in tandem, since a ternary complex could possibly be formed by simultaneous recognition of thrombin. When adapting aptamers to a defined solid phase and to a specific detection technique, appropriate and sometimes profound post-SELEX chemical modifications must be introduced. In the case of microarray, immobilization to the solid support and labeling for detection imply precise chemistries that could sensibly alter the aptamer structure. In fact, since aptamers are evolved in solution, any modification altering the chemical identity of aptamer could affect its folding and consequently its binding to the target: therefore, great care must be taken when developing aptamer-based detection methods in solid phase employing aptamers previously selected in solution. To investigate whether post-SELEX chemical modifications introduced in aptamers would affect thrombin recognition prior to the development of a Sandwich Aptamer Microarray (SAM) system, we adopted these two aptamers and human thrombin as a model system: an extensive analysis on the binary complexes formation has been performed in solution by Electrophoresis Mobility Shift Assay (EMSA) and the verification of the sandwich complex formation in solution by a Supershift assay, incubating simultaneously the two aptamers with the target protein. The validated system was finally applied to the solid phase using an appropriate control and two different protocols for detection. An aptamer-based microarray for thrombin detection has therefore been created, in a sandwich-type format, for recognition of human thrombin with high specificity, using standard microarray slides and a fast and easy protocol. The aptamer-based biosensor could be used to analyze thrombin plasmatic concentration in pathologic conditions, using an easy and reliable protocol.