Complex formation with protamine prolongs the thrombin-inhibiting effect of DNA aptamer in vivo

Antithrombin DNA aptamersRE31 are single-chain oligonucleotides that fold into three-dimensional forms allowing them to bind the enzyme with high affinity and inhibit its activity in vivo. They are rapidly degraded by a nonspecific nuclease, and, to prolong the lifetime of the aptamer DNA in the bloodstream, it is necessary to coat it with a polymer envelope. A new approach to solving this problem based on preparation of DNA-polyelectrolyte complexes with a minimal particle size that can circulate with blood flow. In our experiments, the negatively charged aptamer DNA RE31 was coated step-by-step with positively charged protamine. They had protamine/aptamer ratios of 0.2/1 and 0.4/1 by charge, with particle size being determined by dynamic light scattering. The aptamer DNA-protamine complexes were administered to rats, followed by ex vivo analysis of blood samples. The results showed that prothrombin time (PT) increased by a factor of 5.6-6.7 within 2 h after injection and remained at approximately the same level for 6 h, while injections of pure protamine did not lead to any noticeable change in clotting time. Thus, complexation with protamine proved to prolong the inhibitory activity of the RE31 DNA aptamer.