A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm

Abstract

Thrombin-inhibiting DNA aptamers have already been obtained through the systematic evolution of ligands by exponential enrichment (SELEX). However, SELEX is a method that screens DNA aptamers that bind to their target molecules, and it sometimes fails to screen good inhibitors. Therefore, it is necessary to develop a method of screening DNA aptamers based on their inhibitory effects on the target molecules. We developed a novel method of detecting aptamers using an evolution-mimicking algorithm, and we applied it to the search of new aptamers which inhibit thrombin. First, we randomly designed and synthesized ten 15mer oligonucleotides presumed to form G-quartet structures, and then measured their thrombin-inhibiting activities. The aptamers showing high inhibitory activity were selected, and we shuffled and mutated those sequences in silico to generate 10 new sequences of next-generation aptamers. After repeating the cycle five times, we successfully obtained the same aptamers reported previously, and they showed high inhibitory activity. In addition, we added 8mer oligonucleotides to both the 5′ and the 3′ end of the selected 15mer aptamers, and then repeated the evolution in silico. After two cycles, we were able to obtain aptamers with higher inhibitory activity than that of the 15mer aptamers.

Using photons to manipulate enzyme inhibition by an azobenzene-modified nucleic acid probe

“…Second, even minor modifications to 15Apt can result in significant disruption of interacting configurations (34), which are generally unpredictable without computational simulation or experimental investigation. Thus, modifying the complementary 15Apt sequence with azobenzene is a more feasible approach.…”

Inhibition of Thrombin Activity with DNA-Aptamers

“…We confi rmed that it inhibits thrombin clotting and thrombin stimulation of platelet aggregation. The other aptamer, 31TBA, is based on the 15TBA aptamer sequence sup plemented with 8 nucleotides from the 3' and 5' terminals [7]. Its antithrombin effects were not studied until present.…”

“…The sequence and antithrombin characteristics of 15TBA were described earlier [3,6]. The sequence of 31TBA is also known [7], but its effects on functional activity of thrombin were not studied.…”

Aptamer Sensors Combined with Enzymes for Highly Sensitive Detection

“…We used a 31-mer thrombin-binding aptamer (TBA) that we optimized as the enzyme-inhibiting aptamer (Fig. 4(a)) (Ikebukuro et al 2005b). The TBA forms a G-quadruplex structure that plays an important role in its inhibitory activity.…”

“…Although in vitro selection is a powerful method to obtain aptamers, selected aptamers often require optimization for the best affinity and specificity. We can perform post-optimization of their sequences after SELEX through iterative cycles of mutated DNA synthesis and evaluation of their functions, which would be a cumbersome process for antibodies (Ikebukuro et al, 2005b;Ikebukuro et al, 2006;Knight et al, 2009;Noma et al, 2006b;Savory et al, 2010). The fifth advantage is important so that aptamers can be perceived as unique reagents for analytical applications, not as alternatives to antibodies.…”