[Comparative thermodynamic analysis of thrombin interaction with anti-thrombin aptamers and their heterodimeric construct].

Aptamers interacting selectively with the anion-binding exosites 1 and 2 of thrombin were merged into dimeric oligonucleotide constructs with use of a poly-(dT)-linker of 35 nucleotides (nt) long. Complexes of thrombin with the aptamers and their hetero- and homodimeric constructs were measured using the optical biosensor Biacore-3000. K(D) values measured for the hetero- and homodimeric constructs were correspondingly 25-30- and 2-3-fold lower than those for the primary aptamers. Analysis of temperature dependencies of K(D) values within the temperature interval of 10 degrees C-40 degrees C has shown that the values of enthalpy change deltaH upon formation of complexes of thrombin with the aptamers and the hetrodimeric construct are close. The value of the entropy change deltaS upon complex formation of thrombin with the aptamer heterodimeric construct was 1.5-2-fold higher than deltaS values for the complexes with the aptamers. The complex formation and dissociation rates increased with the elevation of temperature from 10 degrees C to 37 degrees C. However, the dissociation rate for the complex of thrombin with the heterodimeric construct was evidently lower that that for the complexes with the aptamers.

[Photoaptamer heterodimeric constructs as a new approach to enhance the efficiency of formation of photocrosslinking with a target protein].

Using two DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nt in length) to produce aptamer heterodimeric constructs results into affinity enhancement. The apparent dissociation constant (Kd(app)) measured at the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (Kd(app) = 0.2-0.4 nM) which were approximately 30-fold less than for the complexes with the primary aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer used could bind to the exosite 1. The measured value of Kd(app) for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5.3 and 190 nM, respectively). Upon exposure to the UV radiation at 308 nm of the equimolar mixtures of thrombin with the photoaptamer construct, the equal yield of the crosslinked complexes was observed at concentrations which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.

[Aptamers as perspective affine reagents for clinical proteomics].

Application of proteomic results to scientific and medical practice will depend in many respects on progress of affine microchips technologies that determine the continuous search for inexpensive and robust affine reagents alternative to monoclonal antibodies. Among synthetic mimetics of antibodies, the oligonucleotide aptamers are of the greatest interest as affine reagents due to the possibility to automate their selection and due to the low cost of oligonucleotide synthesis. In the review the problems related to the automation and optimization of aptamer selection and to the selection of photoaptamers capable to formation of photo-induced covalent complexes with protein targets have been considered. The existing approaches to the post-selection modification of the aptamers to increase theirs affinity and selectivity to protein targets are discussed.