Comparison of the 'chemical' and 'structural' approaches to the optimization of the thrombin-binding aptamer

Abstract

Noncanonically structured DNA aptamers to thrombin were examined. Two different approaches were used to improve stability, binding affinity and biological activity of a known thrombin-binding aptamer. These approaches are chemical modification and the addition of a duplex module to the aptamer core structure. Several chemically modified aptamers and the duplex-bearing ones were all studied under the same conditions by a set of widely known and some relatively new methods. A number of the thrombin-binding aptamer analogs have demonstrated improved characteristics. Most importantly, the study allowed us to compare directly the two approaches to aptamer optimization and to analyze their relative advantages and disadvantages as well as their potential in drug design and fundamental studies.

Figure 1. Schematic representation of the thrombin-binding aptamer and its chemical modifications.

Figure 2. Evaluation of thrombin/aptamer interactions using PC SW biosensors.

Delta H  =  increment of the effective adlayer thickness. A: Sensorgrams obtained upon aptamer binding with surface-immobilized thrombin. Delta H is normalized to thrombin adlayer effective thickness of 1 nm. B: Comparison of specific and nonspecific binding. Random ON is a G-rich ON of about the same length as TBA, which does not adopt monomolecular G-quadruplex structure under the specified conditions, as was proven by CD and UV-melting studies (Random ON  =  GGGAGGCTGATTCAGG). C: Sensorgrams obtained upon thrombin binding with surface-immobilized biotinylated TBA. Thr  =  thrombin. Delta H is normalized to the effective aptamer adlayer thickness of 0.25 nm. D: Sensorgrams obtained upon thrombin binding with surface-immobilized biotinylated thio-TBA. Delta H is normalized to the aptamer adlayer thickness of 0.5 nm. All experiments were performed in duplicate. Saturation level deviation did not exceed 5%.

Figure 3. Thio-TBA/thrombin MD simulation results.

A: The snapshot taken at 10 ns of RR thio-TBA/thrombin dynamics simulation. The aptamer does not dissociate from the protein, and no significant distortions in GQ structure can be seen. Red dotted lines are H-bonds. B: Plots of the thrombin-aptamer binding energy: the total binding energy (top) and the van-der-Waals contribution (bottom).

Figure 4. Schematic representation of TBA analogs with a duplex module and flanks.

Figure 5. The assembly of dsf-TBA31 and its binding with thrombin.

A: EMSA results illustrating the formation of the intermolecular dsf-TBA31 structure and its complex with thrombin. 1– ssf-TBA31; 2 and 3– dsf-TBA-31 (ssf-TBA-31+ strand 2); 4– strand 2 of dsf-TBA31; 5– ssf-TBA31+ thrombin; 6 and 7– dsf-TBA31+ thrombin. B: PCSW-sensorgrams illustrating dsf-TBA31 binding with thrombin. The sensorgrams were obtained upon aptamer interaction with surface-immobilized protein. Random ON  =  GGGAGGCTGATTCAGG. Delta H  =  increment of the effective adlayer thickness.